Methods for renal function determination

ABSTRACT

A method for determining a glomerular filtration rate (GFR) in a patient includes administering to said patient a compound of Formula I and transdermally measuring spectral energy emitted by the compound of Formula I over a measurement time window. The spectral energy is emitted by the compound of Formula I in response to electromagnetic radiation delivered to the compound of Formula I. The method also includes determining the GFR in said patient based on the measured spectral energy emitted by the compound of Formula I over the measurement time window by fitting an exponential function to the spectral energy as a function of time or a linear function to the log of the spectral energy as a function of time to calculate a rate constant associated with renal clearance over the measurement time window and directly related to the GFR normalized to a body size metric of the patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of and claims priority toU.S. patent application Ser. No. 16/171,695, filed on Oct. 26, 2018,which claims priority to U.S. Provisional Patent Application No.62/577,951, filed on Oct. 27, 2017, the disclosure of each of which ishereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The field of the disclosure generally relates to methods andpharmaceutical compositions comprising pyrazine derivatives to assessthe renal function of a patient in need thereof.

Acute renal failure (ARF) is a common ailment in patients admitted togeneral medical-surgical hospitals. Approximately half of the patientswho develop ARF die either directly from ARF or from complicationsassociated with an underlying medical condition, while survivors facemarked increases in morbidity and prolonged hospitalization. Earlydiagnosis is generally believed to be important because renal failure isoften asymptomatic and typically requires careful tracking of renalfunction markers in the blood. Dynamic monitoring of renal functions ofpatients is desirable in order to minimize the risk of acute renalfailure brought about by various clinical, physiological andpathological conditions. Such dynamic monitoring tends to beparticularly important in the case of critically ill or injured patientsbecause a large percentage of these patients tend to face risk ofmultiple organ failure (MOF) potentially resulting in death. MOF is asequential failure of the lungs, liver and kidneys and is incited by oneor more of acute lung injury (ALI), adult respiratory distress syndrome(ARDS), hypermetabolism, hypotension, persistent inflammatory focus andsepsis syndrome. The common histological features of hypotension andshock leading to MOF generally include tissue necrosis, vascularcongestion, interstitial and cellular edema, hemorrhage andmicrothrombi. These changes generally affect the lungs, liver, kidneys,intestine, adrenal glands, brain and pancreas in descending order offrequency. The transition from early stages of trauma to clinical MOFgenerally corresponds with a particular degree of liver and renalfailure as well as a change in mortality risk from about 30% up to about50%.

Traditionally, renal function of a patient has been determined usingcrude measurements of the patient's urine output and plasma creatininelevels. These values are frequently misleading because such values areaffected by age, state of hydration, renal perfusion, muscle mass,dietary intake, and many other clinical and anthropometric variables. Inaddition, a single value obtained several hours after sampling may bedifficult to correlate with other physiologic events such as bloodpressure, cardiac output, state of hydration and other specific clinicalevents (e.g., hemorrhage, bacteremia, ventilator settings and others).

Chronic Kidney Disease (CKD) is a medical condition characterized in thegradual loss of kidney function over time. It includes conditions thatdamage the kidneys and decrease their ability to properly remove wasteproducts from the blood of an individual. Complications from CKD includehigh blood pressure, anemia (low blood count), weak bones, poornutritional health and nerve damage in addition to an increased risk ofheart disease. According to the National Kidney Foundation,approximately two-thirds of all cases of CKD are caused by diabetes orhypertension. In addition to a family history of kidney disease, otherrisk factors include age, ethnicity, hypertension, and diabetes. Therenal glomerular filtration rate (GFR) is the best test to determine thelevel of kidney function and assess the stage of a patient's CKD.

The GFR is an important test to determine the level of kidney functionwhich determines the state of CKD. As shown in Table 1 and FIG. 28 , thelower the GFR, the more serious the CKD. The GFR can be estimated basedon a blood test measuring the blood creatinine level in combination withother factors. More accurate, and therefore more useful, methods requirethe injection of an endogenous substance into a patient followed bycareful monitoring of urine output over a period of time. These areoften contrast agents (CA) that can cause renal problems on their own.Radioisotopes or iodinated aromatic rings are two common categories ofCAs that are used for GFR determination.

TABLE 1 Stage Description GFR At increased Increase of risk factors(e.g., diabetes, high blood >90 risk pressure, family history, age,ethnicity) 1 Kidney damage with normal kidney function >90 2 Kidneydamage with mild loss of kidney function 60-89 3a Mild to moderate lossof kidney function 44-59 3b Moderate to severe loss of kidney function30-44 4 Severe loss of kidney function 15-29 5 Kidney failure; dialysisrequired <15

Contrast Induced Nephropathy (CIN) is a serious complication connectedto the use of PG radioisotopes or iodinated CAs. It is thought that CINis caused by either renal vasoconstriction or tubular injury caused bythe CA. The definition of CIN varies from study to study but the mostcommon definition, based on the symptoms experienced by the patient,include an increase in serum creatinine by at least 25% above baselineoccurring two to five days after exposure to the CA in the absence ofother causes of acute renal failure. CIN can be fatal for up to 20% ofpatients hospitalized due to these complications. The more severe theCKD, the greater the risk for CIN. Thus, the patients most in need ofaccurate GFR data are most at risk for developing sometimes fatalcomplications in getting that data.

With regard to conventional renal monitoring procedures, anapproximation of a patient's glomerular filtration rate (GFR) can bemade via a 24 hour urine collection procedure that (as the namesuggests) typically requires about 24 hours for urine collection,several more hours for analysis, and a meticulous bedside collectiontechnique. Unfortunately, the undesirably late timing and significantduration of this conventional procedure can reduce the likelihood ofeffectively treating the patient and/or saving the kidney(s). As afurther drawback to this type of procedure, repeat data tends to beequally as cumbersome to obtain as the originally acquired data.

Occasionally, changes in serum creatinine of a patient must be adjustedbased on measurement values such as the patient's urinary electrolytesand osmolarity as well as derived calculations such as “renal failureindex” and/or “fractional excretion of sodium.” Such adjustments ofserum creatinine undesirably tend to require contemporaneous collectionof additional samples of serum and/or urine and, after some delay,further calculations. Frequently, dosing of medication is adjusted forrenal function and thus can be equally as inaccurate, equally delayed,and as difficult to reassess as the measurement values and calculationsupon which the dosing is based. Finally, clinical decisions in thecritically ill population are often equally as important in their timingas they are in their accuracy.

It is known that hydrophilic, anionic substances are generally capableof being excreted by the kidneys. Renal clearance typically occurs viatwo pathways: glomerular filtration and tubular secretion. Tubularsecretion may be characterized as an active transport process, andhence, the substances clearing via this pathway typically exhibitspecific properties with respect to size, charge and lipophilicity.

Most of the substances that pass through the kidneys are filteredthrough the glomerulus (a small intertwined group of capillaries in themalpighian body of the kidney). Examples of exogenous substances capableof clearing the kidney via glomerular filtration (hereinafter referredto as “GFR agents”) are shown in FIG. 1 and include creatinine (1),o-iodohippuran (2), and ^(99m)Tc-DTPA (3). Examples of exogenoussubstances that are capable of undergoing renal clearance via tubularsecretion include ^(99m)Tc-MAG3 (4) and other substances known in theart. ^(99m)Tc-MAG3 (4) is also widely used to assess renal functionthough gamma scintigraphy as well as through renal blood flowmeasurement. As one drawback to the substances illustrated in FIG. 1 ,o-iodohippuran (2), ^(99m)Tc-DTPA (3) and ^(99m)Tc-MAG3 (4) includeradioisotopes to enable the same to be detected. Even if non-radioactiveanalogs (e.g., such as an analog of o-iodohippuran (2)) or othernon-radioactive substances were to be used for renal functionmonitoring, such monitoring would typically require the use ofundesirable ultraviolet radiation for excitation of those substances.

Pyrazine derivatives are known in the art for use in renal monitoring,including those disclosed in U.S. Pat. Nos. 8,155,000, 8,664,392,8,697,033, 8,722,685, 8,778,309, 9,005,581, 9,114,160, 9,283,288,9,376,399, and 9,480,687 which are incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, disclosed herein is a compound of Formula I, wherein eachof

X¹ and X² is independently —CO₂R¹, —CONR¹R², —CO(AA) or —CONH(PS); eachof Y¹ and Y² is independently selected from the group consisting of—NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, (CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a peptide chain comprisingone or more amino acids selected from the group consisting of naturaland unnatural amino acids, linked together by peptide or amide bonds andeach instance of AA may be the same or different than each otherinstance; PS is a sulfated or non-sulfated polysaccharide chaincomprising one or more monosaccharide units connected by glycosidiclinkages; and ‘a’ is a number from 1 to 10, ‘c’ is a number from 1 to100, and each of ‘m’ and ‘n’ are independently a number from 1 to 3.

In another aspect, disclosed herein is a system for determining a GFR ina patient in need thereof. The system comprises a computing device, adisplay device communicatively coupled to said computing device, a powersupply that is operatively coupled to said computing device andmaintains electrical isolation of the system from external powersources, one or more sensor heads operatively coupled to said computingdevice, and at least one tracer agent configured to emit spectral energywhen exposed to electromagnetic radiation. The computing device isconfigured to operate and control said sensor heads, record one or moremeasurements sent from said sensor heads, and calculate the GFR of saidpatient based on said measurements. The one or more sensor headscomprise at least one source of electromagnetic radiation and areconfigured to generate and deliver electromagnetic radiation, detect andmeasure the spectral energy emitted by said tracer agent, and transmitsaid measurement emitted by said tracer agent to said computing device.The tracer agent is configured to be administered to said patient, andemit spectral energy that is detectable by said sensor heads whenexposed to electromagnetic radiation.

In still yet another aspect, disclosed herein is a system fortransdermally determining a body-size normalized GFR in a patient. Thesystem comprises a computing device, a display device communicativelycoupled to said computing device, a power supply that is operativelycoupled to said computing device and maintains electrical isolation ofthe system from external power sources, one or more sensor headsoperatively coupled to said computing device, and at least one traceragent configured to emit spectral energy when exposed to electromagneticradiation. The one or more sensor heads comprise at least one source ofelectromagnetic radiation and are configured to generate and deliverelectromagnetic radiation, detect and measure the spectral energyemitted by said tracer agent, and transmit said measurement emitted bysaid tracer agent to said computing device. The tracer agent isconfigured to be administered to said patient, and emit spectral energythat is detectable by said sensor heads when exposed to electromagneticradiation. The computing device is configured to operate and controlsaid sensor heads, record one or more measurements sent from said sensorheads, determine a decay parameter from the measured spectral energyover a measurement time window, determine a quality metric associatedwith the measured spectral energy over the measurement time window, usethe quality metric to assess whether the decay parameter determinationis sufficiently accurate, and if not, increase the measurement timewindow until the quality metric assessment indicates sufficientaccuracy, convert the decay parameter into a body-size-corrected orvolume of distribution (V_(d)) of the tracer agent normalizedmeasurement of GFR and report the result on the display.

In still yet another aspect, disclosed herein is a method fordetermining a glomerular filtration rate (GFR) in a patient in needthereof. The method comprises administering to said patient a compoundof Formula I, or a pharmaceutically acceptable salt thereof, measuring aconcentration of the compound of Formula I in said patient over ameasurement time window, and determining the GFR in said patient,wherein in the compound of Formula I, each of X¹ and X² is independently

—CO₂R¹, —CONR¹R², —CO(AA) or —CONH(PS); each of Y¹ and Y² isindependently selected from the group consisting of —NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a peptide chain comprisingone or more amino acids selected from the group consisting of naturaland unnatural amino acids, linked together by peptide or amide bonds andeach instance of AA may be the same or different than each otherinstance; PS is a sulfated or non-sulfated polysaccharide chaincomprising one or more monosaccharide units connected by glycosidiclinkages; and ‘a’ is a number from 1 to 10, ‘c’ is a number from 1 to100, and each of ‘m’ and ‘n’ are independently a number from 1 to 3.

In still yet another aspect, disclosed herein is a method of assessingrenal function in a patient. The method comprises administering afluorescent compound, or a pharmaceutically acceptable salt thereof, tosaid patient; exposing said fluorescent compound to electromagneticradiation, thereby causing spectral energy to emanate from saidfluorescent compound; detecting the spectral energy emanated from saidfluorescent compound; and assessing renal function of the patient basedon the detected spectral energy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several known contrast agents for renal functionmonitoring.

FIG. 2 is an illustration of a system for monitoring the GFR in apatient.

FIGS. 3A, 3B, 3C and 3D are graphs of the clearance of MB-102illustrating a two-compartment pharmacokinetic model in four differentpatients having different GFR values ranging from 120 mL/min (3A) to 25mL/min (3D).

FIG. 4 is a graph comparing of the GFR determined using MB-102 comparedto Omnipaque®.

FIG. 5 is a bar graph of the percent recovery of MB-102 from the urineof human patients after 12 hours.

FIG. 6 is a bar graph of the plasma concentration half-life of MB-102 inhuman patients.

FIG. 7 is a graph showing the correlation over time between the plasmaconcentration of MB-102 and the transdermal fluorescence intensity, in apatient with a GFR of 117 mL/min/1.73 m².

FIG. 8 is a graph showing the correlation over time between the plasmaconcentration of MB-102 and the trans-cutaneous fluorescence intensity,in a patient with a GFR of 61 mL/min/1.73 m².

FIG. 9 is a graph showing the correlation over time between the plasmaconcentration of MB-102 and the trans-cutaneous fluorescence intensityin a patient with a GFR of 23 mL/min/1.73 m².

FIG. 10 is a graph correlating the transdermally predicted GFR with theplasma measured GFR determined using MB-102 and normalized to bodysurface area of the subject (outlier exclusion method 1; hybrid offsetmethod).

FIG. 11 is a graph correlating the transdermally predicted GFR with theplasma measured GFR determined using MB-102 and normalized to the volumeof distribution of the tracer agent within the subject (outlierexclusion method 1; hybrid offset method).

FIG. 12 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by Iohexol,Un-Normalized (No outlier exclusion; fixed offset fitting method).

FIG. 13 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by Iohexol,BSA-Normalized (No outlier exclusion; fixed offset fitting method).

FIG. 14 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by Iohexol,V_(d)-Normalized (Method 1) (No outlier exclusion; fixed offset fittingmethod).

FIG. 15 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by MB-102,Un-normalized (No outlier exclusion; fixed offset fitting method).

FIG. 16 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by MB-102,BSA-Normalized (No outlier exclusion; fixed offset fitting method).

FIG. 17 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by MB-102,V_(d)-Normalized, Method 1 (No outlier exclusion; fixed offset fittingmethod).

FIG. 18 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: GFR by MB-102,V_(d)-Normalized, Method 2 (No outlier exclusion; fixed offset fittingmethod).

FIG. 19 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Variable Offset Method (Nooutlier exclusion; GFR determination by MB-102 with V_(d) normalizationmethod 2).

FIG. 20 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Hybrid Offset Method (Nooutlier exclusion; GFR determination by MB-102 with V_(d) normalizationmethod 2).

FIG. 21 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Outlier Exclusion Method 1(Hybrid offset method; GFR determination by MB-102 normalized to BSA).

FIG. 22 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Outlier Exclusion Method 1(Hybrid offset method; GFR determination by MB-102 with V_(d)normalization method 2).

FIG. 23 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Outlier Exclusion Method 2(Hybrid offset method; GFR determination by MB-102 normalized to BSA).

FIG. 24 is a graph correlating the plasma-determined GFR to thetrans-cutaneous fluorescence clearance rate: Outlier Exclusion Method 2(Hybrid offset method; GFR determination by MB-102 with V_(d)normalization method 2).

FIG. 25 is a graph summarizing the optimization of the RDTC transitionfor determining the offset method used in fitting the RDTC.

FIG. 26 is a graph summarizing the optimization of the Outlier ErrorThreshold for the Fluorescence Decay Rate Constant.

FIG. 27 is a graph summarizing optimization of the Outlier ErrorThreshold for plasma-determined GFR.

FIG. 28 is a graphical depiction of the 5 stages of chronic kidneydisease by GFR.

FIG. 29 a is a graph of eGFR vs. plasma PK-determined GFR, normalizedfor subject body surface area (nGFR). Superimposed on the graph is anerror grid, indicating diagnosis accuracy, by number of CKD stages.Measurements falling within a grid with only green sides would becorrectly diagnosed by eGFR. Measurements falling within a grid withboth green and yellow sides would be incorrectly diagnosed by eGFR byone CKD stage. Measurements falling within a grid with both yellow andred sides would be incorrectly diagnosed by eGFR by two CKD stages.

FIG. 29 b is a graph of transdermally determined GFR (tGFR) vs. plasmaPK-determined GFR, normalized for subject body surface area (nGFR).Superimposed on the graph is an error grid, indicating diagnosisaccuracy, by number of CKD stages. Measurements falling within a gridwith only green sides would be correctly diagnosed by tGFR. Measurementsfalling within a grid with both green and yellow sides would beincorrectly diagnosed by tGFR by one CKD stage. Measurements fallingwithin a grid with both yellow and red sides would be incorrectlydiagnosed by tGFR by two CKD stages.

FIG. 29 c is a graph of transdermally determined GFR (tGFR) vs. plasmaPK-determined GFR, normalized for the volume of distribution of thetracer agent within the subject (nGFR). Superimposed on the graph is anerror grid, indicating diagnosis accuracy, by number of CKD stages.Measurements falling within a grid with only green sides would becorrectly diagnosed by tGFR. Measurements falling within a grid withboth green and yellow sides would be incorrectly diagnosed by tGFR byone CKD stage. Measurements falling within a grid with both yellow andred sides would be incorrectly diagnosed by tGFR by two CKD stages.

FIG. 30 is a graph of transdermally-measured GFR automaticallydetermined at two body sites in real-time.

DETAILED DESCRIPTION OF THE INVENTION

All references herein to the “pyrazine”, “pyrazine derivative”,“pyrazine molecule”, “pyrazine compound” or “pyrazine analog” apply toall compounds of Formula I. Additionally each reference to the pyrazineincludes all pharmaceutically acceptable salts thereof unlessspecifically stated otherwise. Salt forms may be charged or uncharged,and may be protonated to form the appropriate cation or deprotonated toform the appropriate anion. All aspects and embodiments disclosed hereinare applicable to compounds of Formula I, and specific examples are onlyillustrative and non-limiting to the scope of the disclosure.

In one aspect, disclosed herein is a pyrazine derivative of Formula I,or a pharmaceutically acceptable salt thereof,

wherein each of X¹ and X² is independently —CO₂R¹, —CONR¹R², —CO(AA) or—CONH(PS); each of Y¹ and Y² is independently selected from the groupconsisting of —NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a peptide chain comprisingone or more amino acids selected from the group consisting of naturaland unnatural amino acids, linked together by peptide or amide bonds andeach instance of AA may be the same or different than each otherinstance; PS is a sulfated or non-sulfated polysaccharide chaincomprising one or more monosaccharide units connected by glycosidiclinkages; and ‘a’ is a number from 0 to 10, ‘c’ is a number from 1 to100, and each of ‘m’ and ‘n’ are independently a number from 1 to 3. Inanother aspect, ‘a’ is a number from 1 to 10. In still yet anotheraspect, ‘a’ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In some aspects, at least one of X¹ and X² is —CO(PS) or —CO(AA). In yetanother aspect, both X¹ and X² are —CO(AA).

(AA) is a peptide chain comprising one or more natural or unnaturalamino acids linked together by peptide or amide bonds. The peptide chain(AA) may be a single amino acid, a homopolypeptide chain or aheteropolypeptide chain, and may be any appropriate length. In someembodiments, the natural or unnatural amino acid is an a-amino acid. Inyet another aspect, the a-amino acid is a D-a-amino acid or an L-a-aminoacid. In a polypeptide chain comprising two or more amino acids, eachamino acid is selected independently of the other(s) in all aspects,including, but not limited to, the structure of the side chain and thestereochemistry. For example, in some embodiments, the peptide chain mayinclude 1 to 100 amino acid(s), 1 to 90 amino acid(s), 1 to 80 aminoacid(s), 1 to 70 amino acid(s), 1 to 60 amino acid(s), 1 to 50 aminoacid(s), 1 to 40 amino acid(s), 1 to 30 amino acid(s), 1 to 20 aminoacid(s), or even 1 to 10 amino acid(s). In some embodiments, the peptidechain may include 1 to 100 a-amino acid(s), 1 to 90 a-amino acid(s), 1to 80 a-amino acid(s), 1 to 70 a-amino acid(s), 1 to 60 a-amino acid(s),1 to 50 a-amino acid(s), 1 to 40 a-amino acid(s), 1 to 30 a-aminoacid(s), 1 to 20 a-amino acid(s), or even 1 to 10 a-amino acid(s). Insome embodiments, the amino acid is selected from the group consistingof D-alanine, D-arginine D-asparagine, D-aspartic acid, D-cysteine,D-glutamic acid, D-glutamine, glycine, D-histidine, D-homoserine,D-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine,D-proline, D-serine, D-threonine, D-tryptophan, D-tyrosine, andD-valine. In some embodiments, the a-amino acids of the peptide chain(AA) are selected from the group consisting of arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, homoserine, lysine,and serine. In some embodiments, the a-amino acids of the peptide chain(AA) are selected from the group consisting of aspartic acid, glutamicacid, homoserine and serine. In some embodiments, the peptide chain (AA)refers to a single amino (e.g., D-aspartic acid or D-serine).

In some embodiments, (AA) is a single amino acid selected from the groupconsisting of the 21 essential amino acids. In other aspects, AA isselected from the group consisting of D-arginine, D-asparagine,D-aspartic acid, D-glutamic acid, D-glutamine, D-histidine,D-homoserine, D-lysine, and D-serine. Preferably, AA is D-aspartic acid,glycine, D-serine, or D-tyrosine. Most preferably, AA is D-serine.

In some embodiments, (AA) is a β-amino acid. Examples of β-amino acidsinclude, but are not limited to, β-phenylalanine, β-alanine,3-amino-3-(3-bromophenyl)propionic acid, 3-aminobutanoic acid,cis-2-amino-3-cyclopentene-1-carboxylic acid,trans-2-amino-3-cyclopentene-1-carboxylic acid, 3-aminoisobutyric acid,3-amino-2-phenylpropionic acid, 3-amino-4-(4-biphenylyl)butyric acid,cis-3-amino-cyclohexanecarboxylic acid,trans-3-amino-cyclohexanecarboxylic acid, 3amino-cyclopentanecarboxylicacid, 3-amino-2-hydroxy-4-phenylbutyric acid,2-(aminomethyl)phenylacetic acid, 3-amino-2-methylpropionic acid,3-amino-4-(2-naphthyl)butyric acid, 3-amino-5-phenylpentanoic acid,3-amino-2-phenylpropionic acid, 4-bromo-β-Phe-OH, 4-chloro-β-Homophe-OH,4-chloro-β-Phe-OH, 2-cyano-β-Homophe-OH, 2-cyano-β-Homophe-OH,4-cyano-β-Homophe-OH, 3-cyano-β-Phe-OH, 4-cyano-β-Phe-OH,3,4-dimethoxy-β-Phe-OH, γ,γ-diphenyβ-Homoala-OH, 4-fluoro-β-Phe-OH,β-Gln-OH, β-Homoala-OH, β-Homoarg-OH, β-Homogln-OH, β-Homoglu-OH,β-Homohyp-OH, β-Homoleu-OH, β-Homolys-OH, β-Homomet-OH,β2-homophenylalanine, β-Homophe-OH, β3-Homopro-OH, β-Homoser-OH,β-Homothr-OH, β-Homotrp-OH, β-Homotrp-OMe, β-Homotyr-OH, β-Leu-OH,β-Leu-OH, β-Lys(Z)—OH, 3-methoxy-β-Phe-OH, 3-methoxy-β-Phe-OH,4-methoxy-β-Phe-OH, 4-methy-β-Homophe-OH, 2-methyl-β-Phe-OH,3-methyl-β-Phe-OH, 4-methyl-β-Phe-OH, β-Phe-OH,4-(4-pyridyl)-β-Homoala-OH, 2-(trifluoromethyl)-β-Homophe-OH,3-(trifluoromethyl)-β-Homophe-OH, 4-(trifluoromethyl)-β-Homophe-OH,2-(trifluoromethyl)-β-Phe-OH, 3-(trifluoromethyl)-β-Phe-OH,4-(trifluoromethyl)-β-Phe-OH, β-Tyr-OH, Ethyl 3-(benzylamino)propionate,β-Ala-OH, 3-(amino)-5-hexenoic acid, 3-(amino)-2-methylpropionic acid,3-(amino)-2-methylpropionic acid, 3-(amino)-4-(2-naphthyl)butyric acid,3,4-difluoro-β-Homophe-OH, γ,γ-diphenyl-β-Homoala-OH,4-fluoro-β-Homophe-OH, β-Gln-OH, β-Homoala-OH, β-Homoarg-OH,β-Homogln-OH, β-Homoglu-OH, β-Homohyp-OH, β-Homoile-OH, β-Homoleu-OH,β-Homolys-OH, β-Homomet-OH, β-Homophe-OH, β3-homoproline, β-Homothr-OH,β-Homotrp-OH, β-Homotyr-OH, β-Leu-OH, 2-methyl-β-Homophe-OH,3-methyl-β-Homophe-OH, β-Phe-OH, 4-(3-pyridyl)-β-Homoala-OH,3-(trifluoromethyl)-β-Homophe-OH, β-Glutamic acid, β-Homoalanine,β-Homoglutamic acid, β-Homoglutamine, β-Homohydroxyproline,β-Homoisoleucine, β-Homoleucine, β-Homomethionine, β-Homophenylalanine,β-Homoproline, β-Homoserine, β-Homothreonine, β-Homotryptophan,β-Homotyrosine, β-Leucine, β-Phenylalanine, Pyrrolidine-3-carboxylicacid and β-Dab-OH.

(PS) is a sulfated or non-sulfated polysaccharide chain including one ormore monosaccharide units connected by glycosidic linkages. Thepolysaccharide chain (PS) may be any appropriate length. For instance,in some embodiments, the polysaccharide chain may include 1 to 100monosaccharide unit(s), 1 to 90 monosaccharide unit(s), 1 to 80monosaccharide unit(s), 1 to 70 monosaccharide unit(s), 1 to 60monosaccharide unit(s), 1 to 50 monosaccharide unit(s), 1 to 40monosaccharide unit(s), 1 to 30 monosaccharide unit(s), 1 to 20monosaccharide unit(s), or even 1 to 10 monosaccharide unit(s). In someembodiments, the polysaccharide chain (PS) is a homopolysaccharide chainconsisting of either pentose or hexose monosaccharide units. In otherembodiments, the polysaccharide chain (PS) is a heteropolysaccharidechain consisting of one or both pentose and hexose monosaccharide units.In some embodiments, the monosaccharide units of the polysaccharidechain (PS) are selected from the group consisting of glucose, fructose,mannose, xylose and ribose. In some embodiments, the polysaccharidechain (PS) refers to a single monosaccharide unit (e.g., either glucoseor fructose). In yet another aspect, the polysaccharide chain is anamino sugar where one or more of the hydroxy groups on the sugar hasbeen replaced by an amine group. The connection to the carbonyl groupcan be either through the amine or a hydroxy group.

In some embodiments, for the pyrazine derivative of Formula I, at leastone of either Y¹ or Y² is

where Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or—SO₂—; and each of R¹ to R² are independently selected from the groupconsisting of H, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; a, c, m and n are as describeelsewhere herein.

In yet another aspect, at least one of Y¹ and Y² is —NR¹R², and R¹ to R²are as described above. In yet another aspect, both Y¹ and Y² are—NR¹R², and R¹ to R² are as described above. Alternatively, R¹ and R²are both independently selected from the group consisting of H,—CH₂(CHOH)_(a)CH₃, —(CH₂)_(a)SO₃H, —(CH₂)_(a)NHSO₃H, and—(CH₂)_(a)PO₃H₂. In yet another aspect, both R¹ and R² are hydrogen.

In any aspect of the pyrazine compound, one or more atoms mayalternatively be substituted with an isotopically labelled atom of thesame element. For example, a hydrogen atom may be isotopically labelledwith deuterium or tritium; a carbon atom may be isotopically labelledwith ¹³C or ¹⁴C; a nitrogen atom may be isotopically labelled with ¹⁴Nor ¹⁵N. An isotopic label may be a stable isotope or may be an unstableisotope (i.e., radioactive). The pyrazine molecule may contain one ormore isotopic labels. The isotopic label may be partial or complete. Forexample, a pyrazine molecule may be labeled with 50% deuterium therebygiving the molecule a signature that can be readily monitored by massspectroscopy or other technique. As another example, the pyrazinemolecule may be labeled with tritium thereby giving the molecule aradioactive signature that can be monitored both in vivo and ex vivousing techniques known in the art.

Pharmaceutically acceptable salts are known in the art. In any aspectherein, the pyrazine may be in the form of a pharmaceutically acceptablesalt. By way of example and not limitation, pharmaceutically acceptablesalts include those as described by Berge, et al. in J. Pharm. Sci.,66(1), 1 (1977), which is incorporated by reference in its entirety forits teachings thereof. The salt may be cationic or anionic. In someembodiments, the counter ion for the pharmaceutically acceptable salt isselected from the group consisting of acetate, benzenesulfonate,benzoate, besylate, bicarbonate, bitartrate, bromide, calcium edetate,camsylate, carbonate, chloride, citrate, dihydrochloride, edetate,edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,pamoate, pantothenate, phosphate, diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, triethiodide, adipate, alginate, aminosalicylate,anhydromethylenecitrate, arecoline, aspartate, bisulfate, butylbromide,camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate,jemisulfate, judrofluoride, judroiodide, methylenebis(salicylate),napadisylate, oxalate, pectinate, persulfate, phenylethylbarbarbiturate,picrate, propionate, thiocyanate, tosylate, undecanoate, benzathine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine,procaine, benethamine, clemizole, diethylamine, piperazine,tromethamine, aluminum, calcium, lithium, magnesium, potassium, sodiumzinc, barium and bismuth. Any functional group in the pyrazinederivative capable of forming a salt may optionally form one usingmethods known in the art. By way of example and not limitation, aminehydrochloride salts may be formed by the addition of hydrochloric acidto the pyrazine. Phosphate salts may be formed by the addition of aphosphate buffer to the pyrazine. Any acid functionality present, suchas a sulfonic acid, a carboxylic acid, or a phosphonic acid, may bedeprotonated with a suitable base and a salt formed. Alternatively, anamine group may be protonated with an appropriate acid to form the aminesalt. The salt form may be singly charged, doubly charged or even triplycharged, and when more than one counter ion is present, each counter ionmay be the same or different than each of the others.

In yet another aspect, disclosed herein is a method for measuring therenal glomerular filtration rate (GFR) in a patient in need thereof. Themethod comprises administering to a patient a pyrazine compound, or apharmaceutically acceptable salt thereof, measuring the transdermalfluorescence in said patient over a period of time, and determining theGFR in said patient. The period of time used to determine a singlemeasurement of GFR is referred to herein as the Measurement Time Window.In many situations it will be clinical useful to have a real-timeassessment of GFR over time. Therefore, in some aspects of thedisclosure, multiple sequential assessments of GFR are provided. In someaspects, the multiple sequential GFR estimates are provided after asingle administration of the tracer agent. The total length of time overwhich GFR measurements are provided after a single injection will bereferred to herein as the Single Injection Reporting Period. In someaspects, there is temporal overlap between the Measurement Time Windows.In such cases, the time interval at which GFR is reported (the ReportingTime Interval) is not necessarily the same as the Measurement TimeWindow. For example, in one embodiment, adjacent Measurement TimeWindows overlap by 50%, and the Reporting Time Interval is half of theMeasurement Time Window. In some aspects, the Measurement Time Windowshave variable length. In a preferred embodiment, if temporally adjacentMeasurement Time Windows are of differing length, then the overlap timeperiod is selected to be 50% of the lesser of the two Measurement TimeWindows. In some aspects, the GFR of a patient is determined using thesystem disclosed elsewhere herein.

In yet another aspect, the Measurement Time Window is automaticallyadjusted according to a metric related to the signal quality (hereafterreferred to as a Quality Metric). The Quality Metric may be based onestimates of the fluorescence signal-to-noise ratio (SNR),signal-to-background ratio (SBR), good-of-fit metrics, correlationcoefficient, or any combination thereof. In one aspect, a line is fittedto the log of the fluorescence intensity vs. time over the MeasurementTime Window (or equivalently, a single exponential is fit to thefluorescence intensify vs. time). The difference between the fitted lineand data (“Fitting Residual”) is used to estimate the “Noise”. In oneaspect, the Noise is the root mean square (RMS) of the Fitting Residual.In another aspect, the Noise is the median absolute deviation (MAD) ofthe Fitting Residual. The “Signal” may be defined as the amplitude ofthe single exponential derived from the fit. In another aspect, theSignal is chosen as the difference between the fitted fluorescence atthe beginning and end of the Measurement Time Window. In another aspect,the pre-injection fluorescence signal level is used to determine a“Background”, and the SBR is computed by dividing the Background intothe Signal level. When using either the SNR or SBR as the QualityMetric, a minimum threshold may be defined and only if the QualityMetric exceeds this threshold will the fit be considered valid for thepurpose of determining GFR. In another aspect, the estimated error ofthe time or rate constant determined by the fit to fluorescence vs timeis used as the Quality Metric. In this case, the fit may be consideredvalid only if the computed Quality Metric is below a predeterminedthreshold value. In some other aspects, the fitted time or rate constantis defined as the Signal and the estimated error from the fit is definedas the Noise, and this version of SNR is used as the Quality Metric. Inanother aspect, a correlation coefficient is used as the Quality Metric.Any of various methods known in the art for computing the correlationcoefficient may be employed, such as Pearson's correlation coefficient,or the concordance correlation coefficient. In yet another aspect, acombination of different Quality Metrics are combined into a singlemetric, or the fitted result is only considered valid for the purpose ofdetermining GFR if all of the selected Quality Metrics are passed.

In another aspect, a minimum Measurement Time Window is defined, and aQuality Metric is used to determine whether to report the GFR, or toextend the length of the Measurement Time Window. In one suchembodiment, the length of the Measurement Time Window is automaticallyincreased until the Quality Metric reaches a threshold, at which pointthe GFR is reported. In another aspect, preliminary fits are used to thetime or rate constant, or predicted GFR, and are used to set theMeasurement Time Window to a predetermined length. In one embodiment,the minimum Measurement time is set to 60 minutes, at which point a fitis performed and a preliminary estimate of GFR is made. If thepreliminary estimate of GFR is equal to above 75 mL/min/1.73 m², thenthe result is reported to the user, and the Measurement Time Window iskept at 60 minutes. However, if the preliminary estimated of GFR isbelow 75 mL/min/1.73 m², then the result is not reported to the user,and the Measurement Time Window is increased to 120 minutes.

In another aspect, the remaining Single Injection Reporting Period isestimated and provided to the user periodically. The basis forestimating the remaining Single Injection Reporting Period may be theSNR, SBR, or estimated fitting error, such as the methods describedabove for determining a Quality Metric, but the Quality Metric used todetermine the Measurement Time Window and the Quality Metric used todetermine the remaining Single Injection Reporting Period may be thesame or different. In some aspects, in addition to the Quality Metric, afitted fluorescence decay time or rate constant is used to estimate theremaining Single Injection Reporting Period. In one embodiment, thefitted fluorescence decay time constant and the SNR are combined topredict the remaining Single Injection Reporting Period. The SNR isscaled to range between minimum and maximum values of 0 and 1, and ismultiplied with the fluorescence decay time constant. The product isthen scaled to predict the Single Injection Reporting Period. Thescaling factor is a calibration factor that is determined throughanalysis of data collected previously on human patients, animals, invitro studies, simulations, or any combination thereof.

In yet another aspect, filtering and/or outlier rejection are applied tothe fluorescence data before fitting within the Measurement Time Window.Examples of appropriate filters include: a boxcar average, an infiniteresponse function filter, a median filter, a trimmed mean filter.Examples of outlier rejection methods include all of the above QualityMetrics described above, but applied to a subset of the Measurement TineWindow.

In some aspects, the Quality Metric is computed from the measuredemission energy of the tracer agent as a function of time over ameasurement time window. The Quality Metric may be used to determinewhether or not to report the computed GFR. In other aspects, the QualityMetric is used to decide whether to expand the measurement time window.For example, the measurement time window may be automatically expandeduntil the quality metric passes a predetermined threshold, at which timethe GFR is reported.

GFR normalized to patient body size is determined by fitting themeasured emission energy of the tracer agent as a function of time overa measurement time window to a decay parameter. In some aspects, thisdecay parameter is the rate constant (or its inverse, referred to as atime constant) from a single exponential fit. In some aspects the offsetof the fitted function is fixed at zero; in other aspects the offset isa variable term in the fit; in yet other aspects, whether the offset isfixed or allowed to vary depends on a preliminary assessment of thedecay parameter. The measurement time window is chosen to begin afterthe tracer agent has equilibrated into the body, during the period whenthe decay of the emission intensity is due to renal clearance of thetracer agent. The fitted rate constant is multiplied by a calibrationslope to determine the GFR normalized to patient body size. Thecalibration slope is determined through analysis of data collectedpreviously on human patients, animals, in vitro studies, simulations orany combination thereof.

Because the physical size of a patient can affect the assessment of thefunctioning of the kidneys, in some aspects, a body-size metric is usedto normalize the GFR calculation to further improve the measurement. Insome aspects, the body-size metric used for normalizing the GFR is bodysurface area (BSA). In other aspects, the body-size metric is the volumeof distribution (V_(d)) of the tracer agent.

The methods and system disclosed herein also permit the real-timemonitoring of GFR in a patient. Additionally, multiple GFR measurementsor determinations can be done with a single administration of a traceragent. In some aspects, a single GFR measurement is determined afteradministration of the tracer agent. In other aspects, multiple GFRmeasurements are determined after administration of the tracer agent,providing a real-time GFR trend. In some such aspects, an estimate isprovided of the time remaining during which the remaining concentrationof tracer agent will be sufficient to continue determining GFR.

In yet another aspect, disclosed herein is a method for measuring therenal glomerular filtration rate (GFR) in a patient in need thereof. Themethod comprises administering to a patient a pyrazine compound, or apharmaceutically acceptable salt thereof, measuring the transdermalfluorescence in said patient over a Measurement Time Window, anddetermining the GFR in said patient. In some aspects, the GFR of apatient is determined using the system disclosed elsewhere herein.

In yet another aspect, disclosed herein is a method for determining theGFR in a patient in need thereof. The method comprises administering tosaid patient a compound of Formula I, or a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable formulation thereof,measuring the concentration of the compound of Formula I in said patientover a Measurement Time Window, and determining the GFR in said patient.

In some aspects and still in reference to the above mentioned method,measuring the concentration of the pyrazine includes monitoring thetransdermal fluorescence in the patient. In yet another aspect,measuring the concentration of the pyrazine includes taking aliquots ofblood from the patient and measuring the concentration of the pyrazineby HPLC or other methods as are known it the art. For example, apyrazine may incorporate a radioisotope that can be quantified. In stillyet another aspect, measuring the concentration of the pyrazine mayincluding collecting the urine of the patient over a period of time todetermine the rate in which the kidneys eliminate the compound from thebody of the patient.

In still yet another aspect and still in reference to the abovementioned method, the concentration of the pyrazine in the patient ismonitored by transdermal fluorescence. This may include contacting amedical device with the skin of the patient wherein said medical deviceis configured to cause a fluorescent reaction in the compound of FormulaI, and detecting said reaction. The medical device may contact the skinof the patient in any suitable location. Specific locations known to besuitable are the sternum, lower sternum, pectoralis major, occipitaltriangle, forehead, chin, upper hip, and lower hip. Other locations on apatient may be used as determined by convenience, medical device design,and/or medical necessity. In some aspects, this method uses the systemdisclosed elsewhere herein.

In one aspect of the above-mentioned method, a display is used to promptthe user to attach the sensor at one or more particular body sites. Inone such embodiment, a touch-screen interface is used, and the user isinstructed to touch a rendition of the body site location at which thesensor was attached, in order to move to a next step in the measurementsetup process. This has the benefit of discouraging placement of thesensor on body sites that are not appropriate or optimal for the GFRdetermination.

In another aspect, the next step is setting the light source outputlevels and the detector gain levels. In one such aspect, the detectorgain levels and light source levels are both initially set to a lowstate and then the light source levels are sequentially increased untila targeted signal level is achieved. In one embodiment, the light sourceis the excitation source for the fluorescent GFR agent, and the sourcedrive current is increased until either a targeted fluorescence signalis achieved or a predefined maximum current is reached. In the case thatthe maximum source current is reached without attaining the desiredfluorescence signal level, the detector gain is then sequentiallyincreased until either the targeted fluorescence signal is achieved, orthe maximum detector gain setting is reached.

In some aspects, measurement of the diffuse reflectance of the skin ismade in addition to measurement of fluorescence of the skin and GFRagent. In such aspects, the diffuse reflectance signal may be used todetermine the optimum source output and detector gain levels. In yetfurther aspects, diffuse reflectance measurements are made within thewavelength bands for excitation and emission of the fluorescent GFRagent. In such aspects, setting of the LED source levels and detectorgains may be performed by using the diffuse reflectance instead of thefluorescence signal levels to guide the settings. In one such aspect,the target levels or the diffuse reflectance signals are between 15% and35% of the signal level at which detector or amplifier saturationeffects are observed. This provides head-room for signal fluctuationsthat may be associated with patient movement or other physiologicalvariation. The described procedures for optimizing the light sourceoutput and/or detection gains have the benefit that they provide a meansof compensating for physiological variations across different patients,or across different body sites on the same patient. In one aspect, aprimary factor that is compensated is the melanin content of the skin.Other physiological factors that may require compensation include bloodcontent, water content, and scattering within the tissue volume that isoptically interrogated by the sensor. In another aspect, if the desiredsignal targets are not attained, the user is prevented from proceedingwith the measurement. In this manner, the reporting of inaccurateresults is prevented.

Once the desired signal levels have been successfully achieved, inanother aspect, a baseline signal is recorded. In one such aspect, thestability of the baseline is assessed, such as by fitting a slope to thesignal over time, and the baseline is not accepted as valid unless theslope over time is below a pre-determined threshold. In some aspects, adisplay instructs the user not to proceed with administration of thetracer agent until a stable baseline has been achieved. In this manner,measurement is prevented if the sensor has not been properly positionedor attached. In addition, the user may be prevented from proceeding witha measurement if the tracer agent from a prior injection has not clearedout of the body yet to a desired degree.

Once a stable baseline is acquired, in another aspect of theabove-mentioned method, the tracer agent is injected into the vascularspace of the patient. The tracer agent administration is automaticallydetected as a rapid increase in the transdermal fluorescence of thepatient as measured by the one or more sensors. A predeterminedthreshold for the rate of change, absolute signal change, or relativesignal change may be employed for this purpose. The automatic agentdetection may be reported to the user on a display device, such as atouch-screen monitor. In another aspect, once the tracer agent isdetected, a further threshold is used to determine if sufficient traceragent is present to initiate a GFR measurement. In one such aspect,measurements of fluorescence (F_(meas)) and diffuse reflectance (DR) arecombined in a manner which reduces the influence of physiologicalvariation on the combined result (herein referred to as the IntrinsicFluorescence or IF), so that, for example, the influence of skin coloron the measurement is compensated for. The sufficiency of the traceragent is then assessed by comparing the IF to a pre-determinedthreshold. In some aspects the IF is determined by using a formula ofthe form:

$\begin{matrix}{{IF} = \frac{F_{meas}}{{DR}_{ex}^{kex}{DR}_{em}^{kem}{DR}_{{em},{filtered}}^{{kem},{filtered}}}} & {{Equation}(1)}\end{matrix}$

where the subscripts on the DR terms refer to measurements collectedwithin the tracer agent excitation (ex) and emission (em) wavelengthbands, with both filtered and un-filtered detectors, and thesuperscripts on the DR terms are calibration coefficients that may bedetermined through analysis of data collected previously on humanpatients, animals, in vitro studies, simulations, or any combinationthereof. In this manner, if insufficient tracer agent has beenadministered for an accurate GFR assessment, the medical professionaladministering the measurement may be provided the opportunity toadminister additional tracer agent, or to discontinue the measurement.

Once the tracer agent has been administered, in another aspect, theequilibration of the tracer agent into the extracellular space ismonitored. In one aspect, the Measurement Time Window does not startuntil it has been determined that equilibration is sufficientlycomplete. A fit to an exponential function may be used to assessequilibration progress. For example, the change in fluorescenceintensity over time may be fit to a single exponential function, andonly once the fitted time constant is stable, is equilibration deemed tobe complete. In one such aspect, a running estimate of when the firstGFR determination will become available is provided to the user. Inanother aspect, the user is prevented from proceeding to the measurementphase until and unless sufficient equilibration has been achieved. Inone such aspect, the equilibration time is compared to a predeterminedthreshold, and if the equilibration time exceeds the threshold, the useris prevented from proceeding with GFR determination. In this manner, ifthe sensor is located in a site that is in poor exchange with thecirculatory system, the assessment of GFR is prevented.

In some aspects, the Reporting Time Interval, Measurement Time Window,and/or Single Injection Reporting Period are based on the specificmedical assessment being performed and may vary accordingly. Forexample, for patients with chronic kidney failure, a single GFRdetermination may be sufficient. However, for patients with or at riskof acute kidney failure, a real-time assessment or GFR trend providesgreat potential benefit. In some aspects said Reporting Time Intervalwill be approximately 15 minutes. In other aspects said Reporting TimeInterval will be approximately 30 minutes, approximately one hour,approximately two hours, approximately three hours, approximately fivehours, approximately eight hours, approximately 10 hours, approximately12 hours, approximately 18 hours, approximately 24 hours, approximately36 hours, approximately 48 hours, approximately 72 hours, approximately96 hours, or approximately 168 hours. In some aspects the Reporting TimeInterval will be between 15 minutes and 168 hours. In some aspects theSingle Injection Reporting Period will be based on the clearancehalf-life of the pyrazine compound. Said clearance half-life can beeither previously determined in said patient, estimated based on themedical condition of said patient, or determined transdermally using themethods described herein. In some aspects said Single InjectionReporting Period is one clearance half-life, two clearance half-lives,three clearance half-lives, four clearance half-lives, five clearancehalf-lives, six clearance half-lives, eight clearance half-lives, or tenclearance half-lives. The maximum Single Injection Reporting Period issuch that the pyrazine is no longer detectable in the blood stream ofsaid patient. “Undetectable” as used herein means that the concentrationof the pyrazine is no longer detectable by the method used to make thedetermination. In some instances, when the detection level of theinstrument makes this an extremely long time period (e.g., over oneweek), “undetectable” means that the concentration level has droppedbelow 0.39% (i.e., eight clearance half-lives). In yet another aspect,the Reporting Time Interval is between approximately 1 and 168 hours andall one hour increments in between.

Likewise, the Measurement Time Window may vary according to the specificmedical needs of the patient and may vary accordingly. In some aspectsit will be approximately 15 minutes. In other aspects said MeasurementTime Window will be approximately 30 minutes, approximately one hour,approximately two hours, approximately three hours, approximately fivehours, approximately eight hours, approximately 10 hours, approximately12 hours, approximately 18 hours, approximately 24 hours, approximately36 hours, approximately 48 hours, approximately 72 hours, approximately96 hours, or approximately 168 hours. In some aspects the MeasurementTime Window will be between 15 minutes and 168 hours. There may be oneor a plurality of Measurement Time Windows during each Single InjectionReporting Period. In some aspects, the Single Injection Reporting Periodis divided into multiple Measurement Time Windows where each MeasurementTime Window is the same. In yet another aspect, the Single InjectionReporting Period is divided into multiple Measurement Time Windows whereeach Measurement Time Windows is selected independently of the othersand may be the same or different than the other Measurement TimeWindows.

The methods and system disclosed herein have the benefit ofautomatically adjusting for skin melanin content, such that the GFRdetermination is accurate across a wide range of skin types and levelsof pigmentation. The Fitzpatrick scale is a numerical classificationscheme for human skin color. It is widely recognized as a useful toolfor dermatological research into human skin pigmentation. Scores rangefrom type I (very fair skin with minimal pigmentation) to type VI(deeply pigmented and dark brown). The system and methods disclosedherein are suitable for use with all six categories of skin pigmentationon the Fitzpatrick scale. Specifically, the systems and methodsdisclosed herein are suitable for use with skin pigmentation of type I,type II, type III, type IV, type V and type VI.

In yet another aspect, the pyrazine is combined with at least onepharmaceutically acceptable excipient. Said pharmaceutically acceptableexcipients are selected from the group consisting of solvents, pHadjusting agents, buffering agents, antioxidants, tonicity modifyingagents, osmotic adjusting agents, preservatives, antibacterial agents,stabilizing agents, viscosity adjusting agents, surfactants andcombinations thereof.

Pharmaceutically acceptable solvents may be aqueous or non-aqueoussolutions, suspensions, emulsions, or appropriate combinations thereof.Non-limiting examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Examples of aqueous carriers arewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media.

By way of example and not limitation, pharmaceutically acceptablebuffers include acetate, benzoate, carbonate, citrate, dihydrogenphosphate, gluconate, glutamate, glycinate, hydrogen phosphate, lactate,phosphate, tartrate, Tris HCl, or combinations thereof having a pHbetween 4 and 9, preferably between 5 and 8, most preferably between 6and 8, very most preferably between 7.0 and 7.5. In yet another aspect,the pH is between 6.7 and 7.7. Other buffers, as are known in the art,may be selected based on the specific salt form of the pyrazinederivative prepared or the specific medical application. A preferredbuffer is phosphate buffered saline at physiological pH (approximately7.2).

Examples of the tonicity modifying agent are glycerol, sorbitol,sucrose, or, preferably, sodium chloride and/or mannitol. Examples ofthe viscosity adjusting agent include bentonite, calcium magnesiumsilicate and the like. Examples of the diluent include ethanol, methanoland the like. Examples of the antimicrobial include benzalkoniumchloride, benzethonium chloride, ethylparaben, methylparaben and thelike. Examples of osmotic adjusting agents include aminoethanol, calciumchloride, choline, dextrose, diethanolamine, lactated Ringer's solution,meglumine, potassium chloride, Ringer's solution, sodium bicarbonate,sodium chloride, sodium lactate, TRIS, or combinations thereof. Theseexamples are for illustration only and are not intended to be exhaustiveor limiting.

Also disclosed herein is a method of assessing the renal function in apatient in need thereof, said method comprises administering a compoundof Formula I, or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable formulation thereof, to a patient, exposingsaid patient to electromagnetic radiation thereby causing spectralenergy to emanate from said compound of Formula I, detecting thespectral energy emanated from the compound, and assessing the renalfunction of the patient based on the detected spectral energy.

In some aspects, the compound of Formula I is not metabolized by thepatient; instead it is entirely eliminated by renal excretion withoutbeing metabolized (e.g., no oxidation, glucuronidation or otherconjugation). In some aspects, at least 95% of the compound of Formula Iis not metabolized by the patient prior to renal excretion. In someaspects, at least 96% of the compound of Formula I is not metabolized bythe patient prior to renal excretion. In some aspects, at least 97% ofthe compound of Formula I is not metabolized by the patient prior torenal excretion. In some aspects, at least 98% of the compound ofFormula I is not metabolized by the patient prior to renal excretion. Insome aspects, at least 99% of the compound of Formula I is notmetabolized by the patient prior to renal excretion. In someembodiments, said compound is entirely eliminated by said patient inless than a predetermined period of time. In some aspects, assessing therenal function in a patient may also include determining the GFR in thepatient.

The pyrazine can be administered by any suitable method. The method willbe based on the medical needs of the patient and selected by the medicalprofessional administering the pyrazine or conducting the procedure.Examples of administration methods include, but are not limited to,transdermal, oral, parenteral, subcutaneous, enteral or intravenousadministration. Preferably the pyrazine compound will be administeredusing intravenous or transdermal methods. In some embodiments, thepyrazine is administered via a single bolus intravenous injection. Inyet another embodiment, the pyrazine is administered by multiple bolusintravenous injections. As used herein, transcutaneous and transdermalboth refer to administration through the skin of a patient and are usedinterchangeably.

As used herein, “enteral administration” refers to any method ofadministration that delivers a medicament directly or indirectly to thepatient using the gastrointestinal tract. Examples of enteraladministration include, but are not limited to, oral, sublingual, buccaland rectal. As used herein, “parenteral administration” refers to anymethod of administration that delivers a medicament directly orindirectly to the patient by injection or infusion. Examples orparenteral administration include, but are not limited to, intravenous,intraarterial, intradermal, transdermal, subcutaneous and intramuscular.

Also disclosed herein is a stable, parenteral composition comprising apyrazine derivative of Formula I and a pharmaceutically acceptablebuffering agent. The composition has a tonicity suitable foradministration to a patient via parenteral administration. The tonicityof the parenteral composition may be adjusted using a tonicity adjustingagent as described elsewhere herein. The composition has a pH suitablefor administration to a patient in need thereof and may be adjustedusing a buffer or other pH adjusting agent as described elsewhereherein. The composition has an osmolarity suitable for administration toa patient in need thereof, and the osmolarity of the composition may beadjusted using an osmolarity adjusting agent as described elsewhereherein. The composition is packaged in a sealed container and subjectedto terminal sterilization to reduce or eliminate the microbiologicalburden of the formulation. The composition is stable against degradationand other adverse chemical reactions, and possesses apharmaceutically-acceptable shelf-life.

“Stable”, as used herein, means remaining in a state or condition thatis suitable for administration to a patient. Formulations according tothe present disclosure are found to be stable when maintained at roomtemperature for at least 12 months, and are generally stable at roomtemperature for 12 to 24 months.

A “sterile” composition, as used herein, means a composition that hasbeen brought to a state of sterility and has not been subsequentlyexposed to microbiological contamination, i.e. the container holding thesterile composition has not been compromised. Sterile compositions aregenerally prepared by pharmaceutical manufacturers in accordance withcurrent Good Manufacturing Practice (“cGMP”) regulations of the U.S.Food and Drug Administration. In some aspects, the composition ispackaged in a heat sterilized container. The container may be anycontainer suitable for use in a medical setting, examples include, butare not limited to, a vial, an ampule, a bag, a bottle and a syringe.

In some embodiments, the composition can take the form of a sterile,ready-to-use formulation for parenteral administration. This avoids theinconvenience of diluting a concentrated parenteral formulation intoinfusion diluents prior to infusion or injection, as well as reducingthe risk of microbiological contamination during aseptic handling andany potential calculation or dilution error. Alternatively, theformulation may be a solid formulation that is diluted prior toadministration to the patient.

The aqueous, sterile pharmaceutical composition disclosed herein issuitable for parenteral administration to a patient in need thereof. Forexample, the composition may be administered in the form of a bolusinjection or intravenous infusion. Suitable routes for parenteraladministration include intravenous, subcutaneous, intradermal,intramuscular, intraarticular, and intrathecal. The ready-to-useformulation disclosed herein is preferably administered by bolusinjection. In some embodiments, the composition is suitable fortransdermal delivery into the epidermis or dermis of a patient.Transdermal delivery methods and devices are known in the art and use avariety of methods to deliver the pharmaceutical composition to thepatient.

The aqueous, sterile pharmaceutical composition is formulated incombination with one or more pharmaceutically acceptable excipients asdiscussed elsewhere herein. The aqueous, sterile pharmaceuticalcomposition is formulated such that it is suitable for administration toa patient in need thereof. The tonicity, osmolarity, viscosity and otherparameters may be adjusted using agents and methods as describedelsewhere herein.

In yet another aspect, disclosed herein is an aqueous, sterilepharmaceutical composition for parental administration. The compositioncomprises from about 0.1 to 50 mg/mL of a pyrazine compound of FormulaI. It also comprises from about 0.01 to 2 M buffering agent as disclosedelsewhere herein. It also comprises from about 0-500 mg/mL of anosmotic-adjusting agent and from about 0-500 mg/mL of atonicity-adjusting agent. The aqueous, sterile pharmaceuticalcomposition may also optionally include one or more additionalpharmaceutically acceptable excipients. Examples of pharmaceuticallyacceptable excipients may be selected from the group consisting ofsolvents, pH adjusting agents, buffering agents, antioxidants, tonicitymodifying agents, osmolarity adjusting agents, preservatives,antibacterial agents, stabilizing agents, viscosity adjusting agents,surfactants and combinations thereof. Specific examples of excipientsare disclosed elsewhere herein.

The pyrazine compound used in the aqueous, sterile pharmaceuticalcomposition is any compound disclosed herein. Specific examples include,but are not limited to, all of the compounds prepared in the Examples.One preferred example is(2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxy-propanoic acid) which is the molecule illustrated in Example12 (also identified as MB-102 or 3,6-Diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide).

The pH of the aqueous, sterile pharmaceutical composition is suitablefor administration to a patient. In some aspects the pH is between 4 and9, preferably between 5 and 8, most preferably between 6 and 8, verymost preferably between 7.0 and 7.5. In yet another aspect, the pH isbetween 6.7 and 7.7. Still more preferably, the pH is approximately 7.2in phosphate buffered saline.

In some aspects, the pyrazine is administered to a patient suspected orknown to have at least one medical issue with their kidneys, and themethods disclosed herein are used to determine the level of renalimpairment or deficiency present in said patient. In some aspects, saidpatient has an estimated GFR (eGFR) or previously determined GFR of lessthan 110, less than 90, less than 60, less than 30, or less than 15. TheeGFR of a patient is determined using standard medical techniques, andsuch methods are known in the art. In some aspects a patient will nothave or be suspected to have medical issues with their kidneys. The GFRmonitoring may be done as part of a general or routine health assessmentof a patient or as a precautionary assessment.

As is known in the art, the rate in which a patient eliminates wastefrom their blood stream (i.e., clearance half-life) is dependent on thehealth and proper functioning of their renal system. “Entirelyeliminated” as used in this context means that the level of theypyrazine in the blood stream has dropped below 0.39% (i.e., eighthalf-lives). The clearance half-life will depend on the GFR of thepatient and slows greatly as the functioning of the renal systemdegrades due to illness, age or other physiological factors. In apatient with no known risk factors associated with CKD, having a normalGFR and/or a normal eGFR, the Single Injection Reporting Period is 24hours. In some aspects, the Single Injection Reporting Period for apatient with a GFR or eGFR below 110 is 24 hours. For a patient with aGFR or eGFR below 90, the Single Injection Reporting Period is 24 hours.For a patient with a GFR or eGFR below 60, the Single InjectionReporting Period is 48 hours. For a patient with a GFR or eGFR below 30,the Single Injection Reporting Period is 48 hours. In some aspects, theSingle Injection Reporting Period for a patient with a GFR or eGFR below110 is equal to eight clearance half-lives. For a patient with a GFR oreGFR below 90, the Single Injection Reporting Period is equal to eightclearance half-lives. For a patient with a GFR or eGFR below 60, theSingle Injection Reporting Period is equal to eight clearancehalf-lives. For a patient with a GFR or eGFR below 30, the SingleInjection Reporting Period is equal to eight clearance half-lives.

Because an increase of protein concentration in the urine of a patientmay suggest some manner of kidney impairment or deficiency, the methodsdisclosed herein are suitable for patients whose urinalysis shows anincrease in protein levels. In some aspects, the patient has anincreased level of protein in their urine as determined by standardmedical tests (e.g., a dipstick test). By way of example and notlimitation, the urinalysis of a patient may show an increase in albumin,an increase in creatinine, an increase in blood urea nitrogen (i.e., theBUN test), or any combination thereof.

Still referring to the above-mentioned method, the pyrazine derivativeis exposed to electromagnetic radiation such as, but not limited to,visible, ultraviolet and/or infrared light. This exposure of thepyrazine to electromagnetic radiation may occur at any appropriate timebut preferably occurs while the pyrazine derivative is located insidethe body of the patient. Due to this exposure of the pyrazine toelectromagnetic radiation, the pyrazine emanates spectral energy (e.g.,visible, ultraviolet and/or infrared light) that may be detected byappropriate detection equipment. The spectral energy emanated from thepyrazine derivative tends to exhibit a wavelength range greater than awavelength range absorbed. By way of example but not limitation, if anembodiment of the pyrazine derivative absorbs light of about 440 nm, thepyrazine derivative may emit light of about 560 nm.

Detection of the pyrazine (or more specifically, the spectral energyemanating therefrom) may be achieved through optical fluorescence,absorbance or light scattering techniques. In some aspects, the spectralenergy is fluorescence. In some embodiments, detection of the emanatedspectral energy may be characterized as a collection of the emanatedspectral energy and the generation of an electrical signal indicative ofthe collected spectral energy. The mechanism(s) utilized to detect thespectral energy from the pyrazine derivative present in the body of apatient may be designed to detect only selected wavelengths (orwavelength ranges) and/or may include one or more appropriate spectralfilters. Various catheters, endoscopes, ear clips, hand bands, headbands, surface coils, finger probes and other medical devices may beutilized to expose the pyrazine derivative to electromagnetic radiationand/or to detect the spectral energy emanating therefrom. The devicethat exposes the pyrazine to electromagnetic radiation and detects thespectral energy emanated therefrom may be the same or different. Thatis, one or two devices may be used. The detection of spectral energy maybe accomplished at one or more times intermittently or may besubstantially continuous.

Renal function, or GFR, of the patient is determined based on thedetected spectral energy. This is achieved by using data indicative ofthe detected spectral energy and generating an intensity/time profileindicative of a clearance of the pyrazine derivative from the body ofthe patient. This profile may be correlated to a physiological orpathological condition. For example, the patient's clearance profilesand/or clearance rates may be compared to known clearance profilesand/or rates to assess the patient's renal function and to diagnose thepatient's physiological condition. In the case of analyzing the presenceof the pyrazine derivative in bodily fluids, concentration/time curvesmay be generated and analyzed (preferably in real time) in order toassess renal function. Alternatively, the patient's clearance profilecan be compared to one or more previously measured clearance profilesfrom the same patient to determine if the kidney function of saidpatient has changed over time. In some aspects, renal functionassessment is done using the system disclosed elsewhere herein.

Physiological function can be assessed by: (1) comparing differences inmanners in which normal and impaired cells or organs eliminate thepyrazine derivative from the bloodstream; (2) measuring a rate ofelimination or accumulation of the pyrazine in the organs or tissues ofa patient; and/or (3) obtaining tomographic images of organs or tissueshaving the pyrazine associated therewith. For example, blood poolclearance may be measured non-invasively from surface capillaries suchas those in an ear lobe or a finger, or it can be measured invasivelyusing an appropriate instrument such as an endovascular catheter.Transdermal fluorescence can also be monitored non-invasively on thebody of said patient. Many locations on the epidermis of a patient maybe suitable for non-invasively monitoring the transdermal fluorescence.The site on the patient is preferably one where vasculature to tissueequilibrium occurs relatively quickly. Examples of suitable sites on apatient include, but are not limited to, the sternum, the lower sternum,pectoralis major, the occipital triangle, the forehead, the chin, theupper hip, and the lower hip. Accumulation of a pyrazine derivativewithin cells of interest can be assessed in a similar fashion.

A modified pulmonary artery catheter may also be utilized to, interalia, make the desired measurements of spectral energy emanating fromthe pyrazine derivative. The ability for a pulmonary catheter to detectspectral energy emanating from said pyrazine is a distinct improvementover current pulmonary artery catheters that measure only intravascularpressures, cardiac output and other derived measures of blood flow.Traditionally, critically ill patients have been managed using only theabove-listed parameters, and their treatment has tended to be dependentupon intermittent blood sampling and testing for assessment of renalfunction. These traditional parameters provide for discontinuous dataand are frequently misleading in many patient populations.

Modification of a standard pulmonary artery catheter only requiresmaking a fiber optic sensor thereof wavelength-specific. Catheters thatincorporate fiber optic technology for measuring mixed venous oxygensaturation exist currently. In one characterization, a modifiedpulmonary artery catheter incorporates a wavelength-specific opticalsensor into a tip of a standard pulmonary artery catheter. Thiswavelength-specific optical sensor is utilized to monitor renalfunction-specific elimination of a designed optically detectablechemical entity such as the pyrazine derivatives disclosed herein. Thus,real-time renal function can be monitored by the disappearance/clearanceof an optically detected compound.

In some aspects, the pyrazine compound is administered to a patientwherein said patient has been previously diagnosed with at least Stage 1CKD. In other aspects, said patient has been previously diagnosed withStage 2 CKD, Stage 3 CKD, Stage 4 CKD or Stage 5 CKD. In yet anotheraspect, the patient has not yet been diagnosed with CKD but has one ormore risk factors associated with CKD. In yet another aspect, thepatient has no known risk factors for CKD.

Administration of the pyrazine compound is done by any suitable methodbased on the medical test being performed and the medical needs of thepatient. Suitable methods are disclosed elsewhere herein. Preferably,the pyrazine is administered by either transdermal or intravenousadministration.

Also disclosed herein is a system for determining the GFR or assessingthe renal function in a patient in need thereof. The system comprises acomputing device, a display device communicatively coupled to saidcomputing device, a power supply that is operatively coupled to saidcomputing device and maintains electrical isolation of the system fromexternal power sources, one or more sensor heads operatively coupled tosaid computing device, and at least one tracer agent configured to emitlight when exposed to electromagnetic radiation. The computing device isconfigured to operate and control the sensor heads, record one or morelight measurements sent from said sensor heads, and calculate the GFR ofsaid patient based on said light measurements.

In some aspects, the one or more sensor heads comprise at least onesource of electromagnetic radiation, generate and deliverelectromagnetic radiation to the skin of said patient, detect andmeasure electromagnetic radiation emitted by said tracer agent, andtransmit said measurement of electromagnetic radiation emitted by saidtracer agent to said computing device. In a system with more than onesensor head, each sensor head may be the same or different and theelectromagnetic radiation emitted therefrom may be the same ordifferent. In some aspects the sensor heads are configured to attach tothe skin of said patient. By way of example and not limitation, in asystem with two sensor heads, one sensor head may emit and monitor onewavelength of electromagnetic radiation while the second sensor head mayemit and monitor a different wavelength. This would enable the data tobe compared to increase the accuracy of the GFR determination and theinformation available to the medical professional administering theassessment. In yet another nonlimiting example, in a system with twosensor heads, the two sensor heads are used to separate the localequilibration kinetics from the terminal phase kinetics. This enables amedical professional to determine when equilibration is complete andreduces artifacts due to local movement of the sensors.

In some aspects, the tracer agent is configured to be administered tosaid patient via intravenous or transdermal administration, beeliminated by only glomerular filtration in the kidneys of said patient,and emit light that is detectable by said sensor heads when exposed toelectromagnetic radiation. In some aspects, the tracer agent is apyrazine compound of Formula I as disclosed elsewhere herein. Preferablythe tracer agent is a compound prepared in one of the Examples. Mostpreferably, the tracer agent is(2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoic acid) (also called MB-102 or3,6-diamino-N²,N⁵-bis(D-serine)-pyrazine-2,5-dicarboxamide). In someaspects, the tracer agent is the pyrazine derivative in a formulationsuitable for administration to a patient in need thereof. Suchformulations are described elsewhere herein.

The system for determining the GFR or assessing the renal function in apatient may be configured to carry out the methods disclosed herein on apatient in need thereof. The computing device in the system may be anystandard computer having all of the capabilities implied therewith,specifically including, but not limited to, a permanent memory, aprocessor capable of complex mathematical calculations, a keyboardand/or a mouse for interacting with the computer, and a displaycommunicatively coupled to the computing device. As such, the permanentmemory of the computing device may store any information, programs anddata necessary to carry out the functions of the system for determiningthe GFR or assessing the renal function in a patient. Such information,programs, and data may be standards and/or controls which may be used tocompare transdermal fluorescence values collected by the one or moresensor heads to known values. In some aspects, the computing device maysave results from a previous assessment or GFR determination in apatient so that results obtained at a later date may be compared. Thiswould permit a medical professional to monitor the health of the kidneysof a patient over time. In some aspects, the computing device is alaptop computer. In some aspects, the display device includes a touchscreen.

Additionally, the computing device is configured to calculate the timeconstant for renal decay over a predetermined period of time. In oneaspect, the transdermal fluorescence data in a patient is collected overa predetermined period of time, and a graph is prepared of time (x-axis)versus fluorescence (y-axis). The rate of decay may be curved or linearand a time constant for the rate of decay is calculated. In one aspect,the rate of decay is linear for a semilog(y) plot. The time constant iscompared to known values thereby determining the GFR in the patient. Insome aspects, the rate of decay corresponds to standard first orderkinetics. In yet another aspect, the rate of decay may exhibit amulti-compartment pharmacokinetic model. FIGS. 3A to 3D illustratetwo-compartment pharmacokinetics by which standard pharmacokineticsoftware is able to determine the time constant for renal decay.

GFR determination is done using linear regression, outlier exclusion,calculation of the correlation coefficient (R²) and standard error ofcalibration and more fully described in the Examples.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. Any aspect orembodiment disclosed herein may be used in combination with any otheraspect or embodiment as would be understood by a person skilled in theart. Other examples are intended to be within the scope of the claims ifthey have structural elements that do not differ from the literallanguage of the claims, or if they include equivalent structuralelements with insubstantial differences from the literal languages ofthe claims.

Example 1 Preparation of3,6-diamino-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (200 mg, 1.01mmol), bis-2-(methoxyethyl)amine (372 mL, 335.5 mg, 2.52 mmol), HOBt.H₂O(459 mg, 3.00 mmol), and EDC.HCl (575 mg, 3.00 mmol) were stirredtogether in DMF (20 mL) for 1 h at room temperature. The mixture wasconcentrated to dryness and the residue was partitioned with EtOAc andwater. The layers were separated and the EtOAc solution was washed withsaturated NaHCO₃ and brine. The solution was dried over anhydrousNa₂SO₄, filtered and concentrated. Purification by radial flashchromatography (SiO₂, 10/1 CHCl₃-MeOH) afforded 228.7 mg (53% yield) ofExample 1 as an orange foam: ¹H NMR (300 MHz, CDCl₃), d 4.92 (s, 4H),3.76 (apparent t, J=5.4 Hz, 4H), 3.70 (apparent t, J=5.6 Hz, 4H), 3.64(apparent t, J=5.4 Hz, 4H), 3.565 (apparent t, J=5.4 Hz), 3.67 (s, 6H),3.28 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 167.6 (s), 145.6 (s), 131.0 (s),72.0 (t), 70.8 (t), 59.2 (q), 49.7 (t), 47.1 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.14min on 30 mm column, (M+H)⁺=429. UV/vis (100 mM in PBS) l_(abs)=394 nm.Fluorescence (100 nm) l_(ex)=394 nm l_(em)=550 nm.

Example 23,6-diamino-N²,N⁵-bis(2,3-dihydroxypropyl)pyrazine-2,5-dicarboxamide

Step1. Synthesis of3,6-diamino-N²,N⁵-bis((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)pyrazine-2,5-dicarboxamide

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (350 mg, 1.77mmol), racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (933 mL, 944mg, 7.20 mmol), HOBt.H₂O (812 mg, 5.3 mmol), and EDC.HCl (1.02 g, 5.32mmol) were stirred together in DMF (20 mL) for 16 h at room temperature.The mixture was concentrated to dryness and the residue was partitionedwith EtOAc and water. The layers were separated and the EtOAc solutionwas washed with saturated NaHCO₃ and brine. The solution was dried overanhydrous Na₂SO₄, filtered and concentrated to afford 665 mg (88% yield)of the bis-amide diastereomeric pair as a yellow solid: ¹NMR (300 MHz,CDCl₃) δ 8.38 (t, J=5.8 Hz, 2H), 6.55 (s, 4H), 4.21 (quintet, J=5.8 Hz,2H), 3.98 (dd, J=8.4 Hz, 6.3 Hz, 2H), 3.65 (dd, J=8.4 Hz, J=5.8 Hz, 2H),3.39 (apparent quartet—diastereotopic mixture, J=5.9 Hz, 4H), 1.35 (s,6H), 1.26 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 165.7 (s), 146.8 (s), 126.8(s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1 (t—diastereotopic pair),27.6 (q), 26.1 (q).

Step 2. The product from Step 1 was dissolved in THF (100 mL) andtreated with 1.0 N HCl (2 mL). After hydrolysis was complete, themixture was treated with K₂CO₃ (1 g) and stirred for 1 h and filteredthrough a plug of C18 with using methanol. The filtrate was concentratedto dryness and the residue was triturated with MeOH (50 mL). The solidswere filtered and discarded and the residue was treated with ether (50mL). The precipitate was collected by filtration and dried at highvacuum. This material was purified by radial flash chromatography toafford 221 mg (36% yield) of Example 2 as an orange solid: ¹NMR (300MHz, DMSO-d₆) δ 8.00 (bm, 6H), 5.39 (bs, 2H), 4.88 (bs, 2H), 3.63-3.71(complex m, 2H), 3.40 (dd, J=11.1, 5.10 Hz, 2H), 3.28 (dd, J=11.1, 6.60Hz, 2H), 2.92 (dd, J=12.6, 3.3 Hz, 2H), 2.65 (dd, J=12.6, 8.4 Hz, 2H).LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=4.13 min on 30 mm column, (M+H)⁺=345. UV/vis (100 mM inH₂O) l_(abs)=432 nm. Fluorescence l_(ex)=432 nm, l_(em)=558 nm.

Example 3(2S,2′S)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoicAcid)

Step 1. Synthesis of Dimethyl2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2′S)-bis(3-(benzyloxy)propanoate)

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24mmol), L-Ser(OBn)-OMe HCl salt (647 mg, 2.64 mmol), HOBt.H₂O (570 mg,3.72 mmol) and EDC.HCl (690 mg, 3.60 mmol) in DMF (25 mL) was treatedwith TEA (2 mL). The resulting mixture was stirred for 16 h andconcentrated. Work up as in Example 1 afforded 370 mg (51% yield) of thebisamide as a bright yellow powder: ¹NMR (300 MHz, CDCl₃): δ 8.47 (d,J=8.74 Hz, 2H), 7.25-7.37 (complex m, 10H), 5.98 (bs, 4H), 4.85 (dt,J=8.7, 3.3 Hz, 2H), 4.56 (ABq, J=12.6, Hz, Dn=11.9 Hz, 4H), 3.99 (onehalf of an ABq of d, J=8.7, 3.3, Dn obscured, 2H), 3.76-3.80 (one halfof an ABq—obscured, 2H), 3.78 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 170.5(s), 165.1 (s), 146.8 (s), 138.7 (s) 128.6 (d), 128.1 (d), 127.8 (d),126.9 (s), 73.5 (t), 69.8 (t), 53.0 (q), 52.9 (q). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.93min on 30 mm column, (M+H)+=581.

Step 2. Synthesis of(2S,2′S)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)-bis(azanediyl))bis(3-(benzyloxy)propanoicAcid

The product from step 1 (370 mg, 0.64 mmol) in THF (10 mL) was treatedwith 1.0 N sodium hydroxide (2.5 mL). After stirring at room temperaturefor 30 min, the reaction was judged complete by TLC. The pH was adjustedto approximately 2 by the addition of 1.0 N HCl and the resultingsolution was extracted (3×) with EtOAc. The layers were combined, driedover sodium sulfate, filtered and concentrated to afford 353 mg (100%yield) of the diacid as an orange foam: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), retention time=4.41 min on 30 mmcolumn, (M+H)⁺=553.

Step 3. To the product from step 2 (353 mg, 0.64 mmol) in methanol (20mL) was added 5% Pd/C (300 mg) and ammonium formate (600 mg). Theresulting reaction was heated at reflux for 2 h. The reaction was cooledto room temperature, filtered through a plug of celite and concentrated.The residue was recrystallized from methanol-ether to provide 191 mg(80% yield) of Example 3 as a yellow foam: ¹NMR (300 MHz, DMSO-d₆) δ8.48 (d, J=6.9 Hz, 2H), 6.72 (bs, 4H), 3.95 (apparent quartet, J=5.1 Hz,2H), 3.60 (apparent ABq of doublets; down-field group centered at 3.71,J=9.9, 5.1 Hz, 2H; up-field group centered at 3.48, J=9.9, 6.3 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ 172.9 (s), 164.9 (s), 147.0 (s), 127.0 (s),62.9 (d), 55.7 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over10 min), single peak retention time=1.45 min on 30 mm column,(M+H)⁺=373. UV/vis (100 mM in PBS) l_(abs)=434 nm. Fluorescencel_(ex)=449 nm, l_(em)=559 nm.

Example 43,6-bis(bis(2-methoxyethyl)amino)-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide bis(TFA) Salt

Step 1. Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic Acid

3,6-Diaminopyrazine-2,5-dicarboxylic acid (499 mg, 2.52 mmol) wasdissolved in 48% hydrobromic acid (10 mL) and cooled to 0° C. in anice-salt bath. To this stirred mixture was added a solution of sodiumnitrite (695 mg, 10.1 mmol) in water (10 mL) dropwise so that thetemperature remains below 5° C. The resulting mixture was stirred for 3h at 5-15° C., during which time the red mixture became a yellowsolution. The yellow solution was poured into a solution of cupricbromide (2.23 g, 10.1 mmol) in water (100 mL) and the resulting mixturewas stirred at room temperature. After an additional 3 h, the aqueousmixture was extracted with EtOAc (3×). The combined extracts were dried(Na₂SO₄), filtered and concentrated to afford 440 mg (54% yield)3,6-dibromopyrazine-2,5-dicarboxylic acid as a pale yellow solid: ¹³CNMR (75 MHz, CDCl₃) δ 164.3 (s), 148.8 (s), 134.9 (s). HPLC (5-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=2.95 min on 250 mm column.

Step 2. Synthesis of3-(Bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

The product from step 1 (440 mg, 1.36 mmol) was dissolved in DMF (25mL), treated with HOBt.H₂O (624 mg, 4.08 mmol), and EDC.HCl (786 mg,4.10 mmol) and stirred for 30 min at room temperature.Bis(2-methoxylethyl)amine (620 mL, 559 mg, 4.20 mmol) was added and theresulting mixture was stirred at room temperature for 16 h andconcentrated. The residue was partitioned with water and EtOAc. TheEtOAc layer was separated and the aqueous was extracted again withEtOAc. The combined organic layers were washed with 0.5 N HCl, saturatedsodium bicarbonate, and brine. The organic layer was dried (Na₂SO₄),filtered and concentrated to afford 214 mg of3-(bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide(26% yield) as a brown oil: LCMS (5-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=3.85 min on 30 mm column,(M+H)⁺=608.

Step 3. To the product from step 2 (116 mg, 0.19 mmol) was addedbis(2-methoxylethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and a “spatulatip” of Pd(PPh₃)₄. The resulting mixture was heated to 140° C. for 2 h.The reaction was cooled and concentrated. The residue was purified byflash chromatography (SiO₂, 10/1 CHCl₃-MeOH). The resulting material waspurified again by reverse phase medium pressure chromatography (C18,10-50% manual gradient acetonitrile in 0.1% TFA) to afford 12 mg (10%yield) of Example 4 as an orange-brown film: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.85min on 250 mm column, (M+H)⁺=661. UV/vis (100 mM in PBS) l_(abs)=434 nm.Fluorescence l_(ex)=449 nm, l_(em)=559 nm.

Example 5 3,6-diamino-N²,N⁵-bis(2-aminoethyl)pyrazine-2,5-dicarboxamidebis(TFA) Salt

Step 1. Synthesis of3,6-diamino-N²,N⁵-bis[2-(tert-butoxycarbonyl)-aminoethyl]pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (500 mg, 2.07mmol), tert-butyl 2-aminoethylcarbamate (673 mg, 4.20 mmol), HOBt.H₂O(836 mg, 5.46 mmol) and EDC.HCl (1.05 g, 5.48 mmol) in DMF (25 mL) wasstirred for 16 h and concentrated. Work up as in Example 1 afforded 770mg (76% yield) of the bisamide as an orange foam: ¹NMR (300 MHz,DMSO-d₆) major conformer, d 8.44 (t, J=5.7 Hz, 2H), 6.90 (t, J=5.7 Hz,2H), 6.48 (bs, 4H), 2.93-3.16 (complex m, 8H), 1.37 (s, 9H), 1.36 (s,9H). ¹³C NMR (75 MHz, DMSO-d₆), conformational isomers d 165.1 (s),155.5 (bs), 155.4 (bs), 146.0 (s), 126.2 (s), 77.7 (bs), 77.5 (bs), 45.2(bt), 44.5 (bt), 28.2 (q).

Step 2. To the product from step 1 (770 mg, 1.60 mmol) in methylenechloride (100 mL) was added TFA (25 mL) and the reaction was stirred atroom temperature for 2 h. The mixture was concentrated and the residuetaken up into methanol (15 mL). Ether (200 mL) was added and the orangesolid precipitate was isolated by filtration and dried at high vacuum toafford 627 mg (77% yield) of Example 5 as an orange powder: ¹NMR (300MHz, DMSO-d₆) δ 8.70 (t, J=6 Hz, 2H), 7.86 (bs, 6H), 6.50 (bs, 4H),3.46-3.58 (m, 4H), 3.26-3.40 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 166.4(s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=3.62min on 30 mm column, (M+H)⁺=283. UV/vis (100 mM in PBS) l_(abs)=435 nm.Fluorescence (100 nM) l_(ex)=449 nm, l_(em)=562 nm.

Example 6 3,6-Diamino-N²,N⁵-bis (D-aspartate)-pyrazine-2,5-dicarboxamide

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis (benzylD-O-benzyl-aspartate)-pyrazine-2,5-dicarboxamide

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (600 mg, 2.48mmol), Asp(OBn)-OMe-p-TosH salt (2.43 g, 5.00 mmol), HOBt.H₂O (919 mg,6.00 mmol) and EDC.HCl (1.14 g, 5.95 mmol) in DMF (50 mL) was treatedwith TEA (4 mL). The resulting mixture was stirred over night at roomtemperature. The reaction mixture was concentrated and the residue waspartitioned with water and EtOAc. The EtOAc layer was separated andwashed successively with saturated sodium bicarbonate, water and brine.The EtOAc solution was dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flash chromatography (SiO₂, 50/1 CHCl₃-MeOH to10/1) to afford 1.15 g of the bis-amide (58% yield) as a yellow foam:¹NMR (500 MHz, CDCl₃) δ 8.61 (d, J=8.4 Hz, 2H), 7.29-7.39 (m, 20H), 5.85(bs, 4H), 5.22 (ABq, J=10.0 Hz, Dn=17.3 Hz, 4H), 5.10 (ABq, J=12.2 Hz,Dn=34.3 Hz, 4H), 5.06-5.09 (obs m, 2H), 3.11 (ABq of d, J=17.0, 5.14 Hz,Dn=77.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 170.7 (s), 170.7 (s), 165.4(s), 147.0 (s), 135.7 (s), 135.6 (s), 129.0 (d), 128.9 (d), 128.8 (d),128.75 (d), 128.7 (d), 126.9 (s), 68.0 (t), 67.3 (t), 49.1 (d), 37.0(t). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=5.97 min on 250 mm column, (M+H)⁺=789.

Step 2. To the product from step 1 (510 mg, 0.65 mmol) was added THF (20mL) and water (10 mL). The stirred mixture was added 10% Pd(C) (500 mg)and ammonium formate (1 g). The resulting mixture was heated to 60° C.for 2 h and allowed to cool to room temperature. The mixture wasfiltered through celite and concentrated. The resulting material waspurified again by reverse phase medium pressure chromatography (C18,10-70% manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54%yield) of Example 6 as an orange solid: ¹NMR (300 MHz, DMSO-d₆) δ 8.62(d, J=8.4 Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J=8.4, 5.4 Hz, 2H),2.74-2.88 (complex m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 172.6 (s), 165.2(s), 147.0 (s), 126.6 (s), 60.8 (t), 49.1 (d). LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.01min on 250 mm column, (M+H)⁺=429. UV/vis (100 mM in PBS) l_(abs)=433 nm.Fluorescence (100 nM) l_(ex)=449 nm, l_(em)=558 nm.

Example 73,6-Diamino-N²,N⁵-bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (77.4 mg, 0.15 mmol) in DMF (5 mL) was addedTEA (151 mg, 1.49 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11-tetraoxatetradecan-14-oate (113 mg, 0.34 mmol) and the reactionwas stirred for 16 h at room temperature. The reaction was concentratedand the residue was purified by medium pressure revered phasechromatography (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63mm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 37.4 mg (35% yield) ofexample 7 as an orange film: ¹NMR (300 MHz, DMSO-d₆) d 8.47 (t, J=5.7Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.20-3.60 (complex m, 36H), 3.47 (s,3H), 3.46 (s, 3H), 2.30 (t, J=6.3 Hz, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6(t), 69.5 (t), 69.4 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS(5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peakretention time=4.01 min on 250 mm column, (M+H)⁺=719, (M+Na)⁺=741.UV/vis (100 mM in PBS) l_(abs)=437 nm. Fluorescence (100 nM) l_(ex)=437nm, l_(em)=559 nm.

Example 83,6-Diamino-N²,N⁵-bis(26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (50.3 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (109 mg, 1.08 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (128 mg, 0.25 mmol) andthe reaction was stirred for 16 h at room temperature. The reaction wasconcentrated and the residue was purified by medium pressure reveredphase chromatography (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals40-63 mm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 87.9 mg (82%yield) of example 8 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.46(t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.16-3.73 (complex m, 74H),2.28-2.32 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆)—multiple conformations—d170.1 (s), 169.9 (s) 169.8 (s), 165.1 (s), 146.0 (s), 126.2 (s). 71.2(t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2(t). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), singlepeak retention time=5.90 min on 250 mm column, (M+H)⁺=1071,(M+2H)²⁺=536. UV/vis (100 mM in PBS) l_(abs)=438 nm. Fluorescence (100nM) l_(ex)=438 nm, l_(em)=560 nm.

Example 93,6-Diamino-N²,N⁵-bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetracontan-41-yl)pyrazine-2,5-dicarboxamide

To a solution of Example 5 (53.1 mg, 0.10 mmol) in DMF (5 mL) was addedTEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl2,5,8,11,14,17,20,23,26, 29,32,35-dodecaoxaoctatriacontan-38-oate (144mg, 0.21 mmol) in DMF (2.0 mL) and the resulting mixture was stirred for16 h thereafter. The reaction was concentrated and the residue waspurified by medium pressure revered phase chromatography (LiChroprepRP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63 mm, ˜70 g, 90/10 to 80/200.1% TFA-ACN) to afford 87.5 mg (61% yield) of example 9 as an orangefilm: ¹NMR (300 MHz, DMSO-d₆) δ 8.48 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4Hz, 2H), 7.80-7.86 (m, 2H), 5.94 (bm, 2H), 3.30-3.60 (complex m, 106H),2.26-2.33 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.2 (s), 165.1 (s),146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t),58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% gradient acetonitrile in 0.1%TFA over 10 min), single peak retention time=5.90 min on 250 mm column,(M+2H)²⁺=712. UV/vis (100 mM in PBS) l_(abs)=449 nm. Fluorescence (100nM) l_(ex)=449 nm, l_(em)=559 nm.

Example 10 Bis(2-(PEG-5000)ethyl)6-(2-(3,6-diamino-5-(2-aminoethylcarbamoyl)pyrazine-2-carboxamido)ethylamino)-6-oxohexane-1,5-diyldicarbamate

A solution of Example 5 (25 mg, 0.049 mmol) in DMF (30 mL) was treatedwith TEA (1 mL) and m-PEG2-NHS (1 g, 0.1 mmol) and the resulting mixturewas stirred for 48 h at room temperature. The mixture was concentratedand the residue was partially purified by gel filtration chromatography(G-25 resin, water). The product was concentrated and further purifiedby reverse phase medium pressure chromatography (C18, 10-70% manualgradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield) ofExample 10 as a tan waxy solid: Maldi MS m/z=11393.

Example 11(R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinicAcid

Step 1. Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine

A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in MeOH (500mL) was treated with NBS (34.3 g, 193.1 mmol), portion-wise, over 1hour. The resulting mixture was stirred for 16 hours thereafter. TLCanalysis at this time shows a small amount of starting materialremaining. Another 1.4 g NBS added and reaction heated to 50° C. for 2hours. The mixture was then cooled to 38° C. and treated with NCS (25.8g, 193.1 mmol). The reaction mixture was heated to 50° C. for 16 hoursthereafter. The mixture was then cooled to room temperature and treatedwith water (500 mL). The precipitate was collected by filtration anddried in a vacuum desiccator to afford 45.4 g (97% yield) of2-amino-5-bromo-3,6-dichloropyrazine as a white solid: ¹³C NMR (75 MHz,CDCl₃) δ 149.9 (s), 145.6 (s), 129.6 (s), 121.5 (s). LCMS (15-95%gradient acetonitrile in 0.1% TFA over 10 min), single peak retentiontime=4.51 min on 30 mm column, (M+H)⁺=244, (M+H+ACN)⁺=285.

Step 2. Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile

A mixture of CuCN (8.62 g, 96.3 mmol) and NaCN (4.72 g, 96.3 mmol) washeated under high vacuum to 90° C. The resulting mixture was subjectedto three Argon/Vacuum cycles and placed under a final positive pressureof Argon. The mixture was allowed to cool to room temperature and DMF(150 mL) was added. The heterogeneous mixture was heated to 130° C. for2.5 hours. To the resulting homogeneous mixture of sodium dicyanocupratewas added a solution of the product from step 1 (15.6 g, 64.2 mmol)dissolved in DMF (150 mL), dropwise, over 1 hour. The temperature wasgradually raised to 150° C. and the resulting mixture was stirred atthis temperature for 10 hours thereafter. The reaction was then allowedto cool to room temperature and poured into water (1 L). The resultingmixture was extracted with EtOAc (3×) and the combined extracts werefiltered to remove a flocculent dark solid, washed with brine, dried(Na₂SO₄), filtered again and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 3/1) to afford 6.70 g (55%yield) of the nitrile product as a tan solid: ¹³C NMR (75 MHz, CDCl₃) δ153.9 (s), 149.1 (s), 131.7 (s), 115.4 (s), 111.0 (s). GCMS (Inj.temperature=280° C., 1.0 mL/min helium flow rate, temperature program:100° C. (2 min hold), ramp to 300° C. @ 10° C./min (2 min hold), majorpeak retention time=16.556 min, m/z (EI)=188, 190.

Step 3. Synthesis of5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyra-zine-2-carbonitrile

To the product from step 2 (1.00 g, 5.29 mmol) in ACN (20 mL) was addedbis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and the reactionmixture was heated to 70° C. for 16 hours thereafter. The reaction wascooled and concentrated. The residue was partitioned with EtOAc andwater. The organic layer was separated and the aqueous was extractedagain with EtOAc. The combined organic extracts were washed with brine,dried (Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 10/1 hexanes-EtOAc to 1/1) afforded 950 mg (63%yield) of the desired adduct as a yellow solid: ¹NMR (300 MHz, CDCl₃) δ7.47 (bs, 2H), 3.77 (t, J=5.7 Hz, 4H), 3.52 (t, J=5.4 Hz, 4H), 3.25 (s,6H). ¹³C NMR (75 MHz, CDCl₃) δ 154.7 (s), 152.0 (s), 120.9 (s), 119.5(s), 95.8 (s), 71.0 (t), 59.1 (q), 50.0 (t). LCMS (50-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.91min on 250 mm column, (M+H)⁺=286, (M+Na)⁺=308, (M+Na+ACN)⁺=349.

Step 4. Synthesis of3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile

To the product from step 3 (1.39 g, 4.88 mmol) in 48% hydrobromic acid(20 mL) at 0° C. (ice-salt bath), was added a solution of sodium nitrite(673 mg, 9.75 mmol) in water (10 mL) dropwise over 30 min. The resultingmixture was stirred at 0˜5° C. for 1 h and poured into a stirredsolution of CuBr₂ (1.64 g, 7.34 mmol) in water (100 mL). The resultingmixture was stirred for 16 h at room temperature thereafter. The mixturewas extracted with EtOAc (3×). The combined organic layers were dried(Na₂SO₄), filtered and concentrated. Purification by flash columnchromatography (SiO₂, 50/1 CHCl3-MeOH) afforded 1.00 g (58% yield) ofthe bromide as an orange-brown solid: ¹NMR (300 MHz, CDCl₃) δ 3.99 (t,J=5.4 Hz, 4H), 3.64 (t, J=5.4 Hz, 4H), 3.35 (s, 6H). ¹³C NMR (75 MHz,CDCl₃) δ 152.8 (s), 140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4(t), 59.1 (t), 50.5 (q). LCMS (50-95% gradient acetonitrile in 0.1% TFAover 10 min), single peak retention time=4.55 min on 250 mm column,(M+H)⁺=349, 351.

Step 5. Synthesis of3-(bis(2-methoxyethyl)amino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile

A mixture of the product from step 4 (1.0 g, 2.87 mmol), 2-furanboronicacid (643 mg, 5.75 mmol), Cs₂CO₃ (3.31 g, 10.2 mmol), TFP (35 mol %, 236mg, 1.02 mmol), and Pd₂dba₃-CHCl₃ (5 mol %, 10 mol % Pd, 150 mg) wassubjected to 3 vacuum/Argon cycles and placed under a positive pressureof Argon. Anhydrous dioxane (50 mL) was added and the reaction mixturewas heated to 75° C. for 16 h thereafter. The reaction mixture wascooled to room temperature, diluted with EtOAc (100 mL) and filteredthrough a medium frit. Concentration and purification of the residue byflash chromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded the 757 mg of thefuran adduct (78% yield) as a tan powder: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=6.41min on 250 mm column, (M+H)⁺=337.

Step 6. Synthesis of6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxylic Acid

To a well stirred mixture of ACN (11 mL), CCl4 (7 mL), and water (11 mL)were added sodium periodate (1.07 g, 5.00 mmol) and RuO₂.H₂O (13.3 mg,0.10 mmol), sequentially. The resulting mixture was stirred vigorouslyat room temperature for 30 min and treated with sodium bicarbonate (2.10g, 25.0 mmol) followed by water (5 mL). Vigorous stirring for another 15minutes was followed by the addition of a solution of the product fromStep 5 (276 mg, 0.82 mmol) dissolved in ACN (1 mL). The green mixturewas stirred at room temperature for 5.5 h. The mixture was transferredto a separatory funnel and extracted with EtOAc. The aqueous layer wasadjusted to pH˜3.5 and extracted again with EtOAc (2×). The combinedextracts were washed with 20% sodium bisulfite and brine and dried(Na₂SO₄). Filtration and concentration afforded 140 mg (54% yield) ofcarboxylic acid as a pale yellow solid: LCMS (5-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=5.05min on 250 mm column, (M+H)⁺=315.

Step 7. Synthesis of (R)-dibenzyl2-(6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamido)succinate

A mixture of the product from step 6 (140 mg, 0.45 mmol), EDC.HCl (128mg, 0.67 mmol) and HOBt.H₂O (102 mg, 0.67 mmol) in anhydrous DMF (25 mL)was stirred together at room temperature for 30 min. To this stirredmixture was added (R)-dibenzyl 2-aminosuccinate p-TsOH salt (213 mg,0.44 mmol) followed by TEA (1 mL). The resulting mixture was stirred for16 h thereafter. The reaction mixture was concentrated and partitionedwith EtOAc and saturated sodium bicarbonate solution. The EtOAc layerwas separated and washed with saturated sodium bicarbonate and brine,dried (Na₂SO₄), filtered and concentrated to afford 240 mg (88% yield)of the pyrazine amide as an orange foam: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=8.76min on 250 mm column, (M+H)⁺=610, (M+Na)⁺=632.

Step 8. (R)-dibenzyl2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinate

To the product from step 7 (240 mg, 0.39 mmol) was added morpholine (5mL). The reaction mixture was heated to 70° C. for 2 h. The mixture wascooled and concentrated. The residue was partitioned with EtOAc andwater. The EtOAc layer was separated and washed with saturated sodiumbicarbonate and brine. The EtOAc layer was dried (Na₂SO₄), filtered andconcentrated. Purification by flash column chromatography (SiO₂, 3:1 to1:1 hexanes-EtOAc) afforded 199 mg (75% yield) of the morpholine adductas an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over10 min), single peak retention time=8.76 min on 250 mm column,(M+H)⁺=661, (M+Na)⁺=683.

Step 9. Synthesis of Example 11

The dibenzyl ester (115 mg, 0.17 mmol) in THF (10 mL) was added 1.0 Nsodium hydroxide (4 mL). The mixture was stirred for 1 h at roomtemperature. The pH was adjusted to ˜2 with 1.0 N HCl and the solutionwas concentrated. Purification of the residue by medium pressurereversed phase chromatography (LiChroprep RP-18 Lobar (B) 25×310 mm—EMDchemicals 40-63 mm, ˜70 g, 90/10 to 50/50 0.1% TFA-ACN) afforded 32 mg(27% yield) of example 11 as an orange solid: LCMS (15-95% gradientacetonitrile in 0.1% TFA over 10 min), single peak retention time=4.47min on 250 mm column, (M+H)⁺=481. UV/vis (100 mM in PBS) l_(abs)=438 nm.Fluorescence (100 nM) l_(ex)=449 nm, l_(em)=570 nm.

Example 12(2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoicAcid) (“D-Serine Isomer” or “MB-102”)

Step 1: Formation of Dibenzyl 2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2′R)-bis(3-hydroxypropanoate

A 500 mL round-bottom flask equipped with a Claisen adapter and anaddition funnel was charged with D-serine benzyl ester hydrochloride(24.33 g, 105.0 mmol), and anhydrous DMF (300 mL) was added by cannula.The solution was cooled in an ice-bath and stirred for 15 min under N₂atmosphere. DIPEA (19.16 mL, 110.0 mmol) was added dropwise via additionfunnel over a 30 min period, and after a further 30 min, the coolingbath was removed and the diacid (9.91 g, 50.0 mmol) was added in oneportion. The brick-red suspension was stirred for 30 min and HOBt.H₂O(17.61 g, 115.0 mmol) was added in one portion. After 15 min, thereaction flask was cooled in an ice-bath, and EDC.HCl (22.05 g, 115.0mmol) was added in portions over 15 minutes. The resulting suspensionwas slowly allowed to warm to room temperature and stirred overnight(ca. 17 h) under N₂.

The dark solution was concentrated to a syrupy residue under high vacuum(bath temp 60° C.) that was partitioned between EtOAc and milli-Q H₂O(400 mL each). The layers were separated, and the aqueous layer wasextracted with EtOAc (3×200 mL). The combined EtOAc extracts weresuccessively washed with 0.50 M KHSO₄, saturated NaHCO₃, H₂O, and brine(250 mL each). Removal of the solvent under reduced pressure gave 23.7 gof an orange solid. The crude product was purified by flashchromatography over silica gel using a CHCl₃:MeOH gradient to give thebisamide (19.6 g, 71%) as an orange solid: R_(f) 0.45 [CHCl₃:MeOH (9:1,v/v)]. ¹H NMR (DMSO-d₆) δ 8.56 (d, J=8.0 Hz, 2H, exchangeable with D₂O),7.40-7.33 (m, 10H), 6.76 (s, 4H, exchangeable with D₂O), 5.37 (t, J=5.5Hz, 2H), 5.20 (m, 4H), 4.66-4.63 (dt, J=8.0, 4.0 Hz, 2H), 3.97-3.93 (m,2H), 3.81-3.77 (m, 2H). ¹³C NMR (DMSO-d₆) δ 170.1, 164.9, 146.4, 135.8,128.4, 128.0, 127.6, 125.9, 66.2, 61.1, 54.4. RP-LC/MS (ESI) m/z 553.3(M+H)+ (tR=4.44 min, 5-95% B/6 min). Anal. Calcd for C₂₆H₂₈N₆O₈: C,56.52; H, 5.11; N, 15.21. Found: C, 56.39; H, 5.11; N, 14.99.

Step 2. Formation of(2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoicAcid)

The bisamide (7.74 g, 14.0 mmol) was hydrogenated in the presence of 10%Pd/C (0.774 g) in EtOH:H₂O (560 mL; 3:1, v/v). The reaction mixture waspurged with argon and stirred under hydrogen atmosphere (slow bubbling)at room temperature for 5.5 h. The reaction mixture was again purgedwith Ar, and the catalyst was removed by filtration over Celite. The bedwas washed with EtOH:H2O (400 mL; 1:1, v/v), and the combined filtrateswere concentrated in vacuo. The product was dried under high vacuum. Theresidue was triturated with CH₃CN to give the D-serine isomer (4.89 g,94%) as an orange powder. ¹H NMR (DMSO-d₆) δ 8.46 (d, J=8.3 Hz, 2H,exchangeable with D₂O), 6.78 (br s, 4H, exchangeable with D₂O),4.48-4.45 (dt, J=8.1, 3.9 Hz, 2H), 3.88 (dd, J=11.1, 3.9 Hz, 2H), 3.74(dd, J=11.1, 3.7 Hz, 2H). ¹³C NMR (DMSO-d₆) δ 171.6, 164.7, 146.4,125.9, 61.2, 54.3. RP-LC/MS (ESI) m/z 373.2 (M+H)+ (tR=2.86 min, 5_95%B/6 min). Anal. Calcd for C₁₂H₁₆N₆O₈: C, 38.71; H, 4.33; N, 22.57.Found: C, 38.44; H, 4.51: N, 22.33.

Example 13(2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))Dipropionic Acid (“D-Alanine Isomer”)

Step 1. Formation of Diethyl 2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2R,2′R)-dipropionate

Under an inert atmosphere, a flame dried round-bottom flask (100 mL)equipped with a magnetic stir bar was charged with3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), D-alanine ethyl esterhydrochloride (1.86 g), EDC.HCl (2.70 g), HOBt.H₂O (2.65 g), and Et₃N(2.0 mL) in DMF (anhydrous, 80 mL). Volatiles were removed under reducedpressure at 50° C. to generate a dark semi-solid. After cooling,acetonitrile (˜100 mL) was added and solution allowed to stand for aboutan hour. A red precipitate was isolated by centrifugation, washed withEtOAc and dried. Total weight 1.30 gm of diester (3.06 mmol, 60.6%isolated yield). This material (1.3 g) was taken forward without furtherpurification.

Step 2. Formation of (2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl)) Dipropionic Acid

The diester from Step 1 (1.0 g) and LiOH (4 equivs.) in THF/water werecombined and stirred at ambient temperature for several hours. HPLCindicated complete hydrolysis. The pH was made acidic by addition ofTFA, and the reaction mixture allowed to stand overnight at ambienttemperature. The diacid was obtained by purification of the reactionmixture by prep RPHPLC. Program: 99:1 A:B for 5 minutes then 5:95 A:B at27 minutes @ 50 mL/min. Lambda UV 264 nm and fluorescence; λ_(x)=440 nm,λ_(m)=565 nm. Fractions containing desired product were combined andlyophilized (˜85 C, 15 mtorr) to obtain an orange solid (0.821 g, 2.41mmol, 95.6% isolated yield. M/z 341.13. Proton and carbon NMR wereconsistent with the proposed structure.

Example 143,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionicAcid (“β-Alanine Isomer”)

Step 1. Formation of Dibenzyl 3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionate

Under an inert atmosphere, a flame dried round-bottom flask (100 mL) wascharged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.30 g), benzyl3-aminopropanoate p-toluene sulfonate (1.08 g), EDC.HCl (0.590 g),HOBt.H₂O (0.582 g), and Et₃N (1.50 g) in DMF (anhydrous, 40). Thereaction mixture was stirred overnight at ambient temperature andconcentrated in vacuo to about 10 mL. The remaining DMF was removed bytoluene azeotrope. The reaction mixture was partitioned between EtOAc(3×125 mL) and saturated NaHCO₃ (3×100 mL). The organic layers werecombined and washed with citric acid (10% aqueous, 100 mL) and brine(100 mL). The organic layer was removed, dried (Na₂SO₄ anhydrous) andconcentrated in vacuo to give a crystalline solid, 0.58 g. TLC (silicaon glass, 1:1 EtOAc:hexanes) R_(f)=0.22. The product was purified viaflash chromatography over silica gel to give 0.49 g of product. Massspectrum (ES+) 521.36 (100%), 522.42 (30%), 523.34 (approx. 6%). NMR, ¹H(DMSO-d₆), 400 MHz: 2.55 (4H, m), 3.41 (4H, m) 5.01 (4H, s), 6.44 (4H,s), 7.21 (10H, m), 8.41 (2H, m); ¹³C (DMSO-d₆): 34.18, 35.33, 66.19,126.74, 128.52, 128.92, 136.56, 146.75, 165.63, 171.90.

Step 2. Formation of 3,3′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))dipropionic Acid

The dibenzyl ester in Step 1 (0.92 g) was combined with EtOH (abs., 75mL) and transferred to a Fischer-Porter pressure bottle (6 oz) equippedwith inlet and outlet valves, a pressure gauge (0-100 psig) and a Tefloncoated magnetic stir bar. Water (25 mL) and 10% Pd on carbon (0.2 g,Degussa/Aldrich wet) were added, and the reaction vessel sealed.Following three vacuum/Ar cycles, H₂(g) was introduced from a lecturebottle at 10 psig to a vigorously stirred solution. After 3.5 hours, thereaction was filtered through a pad of celite and the resultingcelite/catalyst bed rinsed with about 500 mL 1:1 EtOH:H₂O to obtain asolution that was concentrated in vacuo. 0.424 g of a solid was isolated(70.5% isolated yield). HPLC/MS gave only a single peak at 9.3 minutes.Mass spectrum (ES+) 341.32 (100%), 342.37 (30%), 344.29 (18%), 270.30(62%). NMR, ¹H (DMSO-d₆), 400 MHz: 2.54 (2H, m), 3.42 (2H, m), 6.52 (2H,s), 7.21 (4H, m), 8.38 (2H, m), 11.9 (2H, bs); ¹³C (DMSO-d₆): 34.20,35.33, 126.77, 146.75, 165.55, 173.57.

Example 152,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic Acid(“Glycine Isomer”)

Step 1. Formation of Diethyl 2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetate

A round-bottom flask (300 mL) equipped with a magnetic stir bar wascharged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (5.0 g), ethylglycinate hydrochloride (5.04 g), EDC.HCl (8.1 g), HOBt.H₂O (8.0 g), andDIPEA (5.9 g) in DMF (anhydrous, 200 mL). A dry argon atmosphere wasmaintained throughout the course of the reaction. The pyrazine wascombined with glycinate, and DMF was added with stirring, under an inertatmosphere. To this was added base and HOBt. After about 15 minutes, EDCwas added portion-wise over 45 min, and the reaction stirred at ambienttemperature under Ar overnight. The reaction mixture was concentrated invacuo until a viscous, semi-solid was obtained. The semi-solid wastreated with toluene (ca. 30 mL) and volatiles removed in vacuo. Aftercooling a solid formed. The crude product was dissolved in 500 mL EtOAcand mixed until two layers formed. The solution was washed with brineand saturated NaHCO₃ and the aqueous layer was removed. The water layerwas washed (2× EtOAc, 150 mL), and the organic layers combined. Theorganic layer was washed with aqueous NaHSO₄, saturated brine, driedover Na₂SO₄, and concentrated to give an solid. Isolated yield: 5.46 g.HPLC analysis 96.9%. M/z 369.2. ¹H and ¹³C NMR consistent with proposedstructure. This product was taken forward without further purification.

Step 2. Formation of 2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))diacetic Acid

The crude product from Step 1 (700 mg) was dissolved in 40 mL THF with10 mL water (DI). LiOH (4.2 equivalents) was added, and the mixturestirred overnight at ambient temperature under an inert atmosphere. HPLCanalysis indicated complete conversion to desired diacid (M/z=313.3).The reaction mixture was centrifuged (3000 rpm for 3 minutes), and thesupernatant checked by HPLC and discarded. The remaining solid wasconverted to the di-sodium salt by treating with NaOH (6.25 N, 2equivalents), and the resulting solution filtered (0.22 micron). Thesolution was lyophilized to give a solid that was >95% pure by HPLC. Thedi-sodium salt was converted to diacid by adding slightly more than twoequivalents of TFA followed by reverse phase preparative column. Protonand carbon NMR were consistent with proposed structure.

Example 16(2S,2′S)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))Dipropionic Acid (“L-Alanine Isomer”)

Step 1. Formation of Diethyl2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))(2S,2′S)-dipropionate

Under an inert atmosphere, a flame dried round-bottom flask (100 mL) wascharged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), ethylL-alaninate hydrochloride (1.86 g), EDC.HCl (2.70 g), HOBt.H₂O (2.65 g)in DMF (anhydrous, 80 mL). Triethylamine was added (1.50 g). After 16hours at ambient temperature, the reaction volatiles were removed invacuo. A semi-solid was isolated. Water was added (70 mL) and themixture allowed to stand for about an hour. During this time aprecipitate formed so the mixture was centrifuged, and a solid isolatedthat was air dried overnight. This material was dissolved in EtOAc andwashed with water, citric acid and saturated sodium bicarbonate. Theorganic layer dried (anhydrous sodium sulfate) and concentrated in vacuoto give a solid product (1.38 g). HPLC purity>95% purity. The crudeproduct was used in the next step without further purification.

Step 2. Formation of(2S,2′S)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis (azanediyl))Dipropionic Acid

The crude product from Step 1 (1.0 g) was dissolved in THF (30 mL), andLiOH H₂O (4 equiv.) dissolved in water (10 mL) was added at ambienttemperature. After an hour, the volatiles were removed in vacuo. Theproduct was purified by preparative reverse phase HPLC and lyophilizedto obtain a solid with >95% purity of desired diacid product. Proton andcarbon NMR were consistent with proposed structure.

Example 172,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoicAcid) (“Dimethyl Glycine Isomer”)

Step 1. Formation of Diethyl2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoate)

Under an inert atmosphere, a flame dried round-bottom flask (100 mL) wascharged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (1.0 g), ethylgem-dimethyl 3-amino propanoic acid hydrochloride (1.86 g), EDC.HCl(2.70 g), HOBt.H₂O (2.65 g) in DMF (anhydrous, 80 mL). The reaction wasinitiated by addition of triethylamine (1.50 g) and maintained atambient temperature for 72 hr. Volatiles were removed in vacuo. A darkviscous liquid was isolated. After cooling, it taken up in acetonitrile(about 100 mL) and allowed to stand for about an hour. A precipitateformed that was isolated by centrifugation and dried to obtain 1.30 g ofthe di-ethyl ester (61%) have a purity by RPHPLC>95%. This crude productwas used directly in the next step.

Step 2. Formation of 2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(2-methylpropanoic Acid)

The crude product from Step 1 (1.0 g) was dissolved in THF:water (40mL:5 mL). To this was added LiOH in water (2.5 equivalents in 0.5 mL DIwater). Another two equivalents of LiOH was added to the reactionmixture and the reaction allowed to proceed overnight at ambienttemperature. Upon completion the reaction mixture was acidified with TFAuntil pH of about 4 has been reached. The product was isolated bypreparative RPHPLC. M/z 369.13. Proton and carbon NMR were consistentwith proposed structure.

Example 183,6-diamino-N²,N⁵-bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide

A round bottom flask (100 mL) equipped with a magnetic stir bar wascharged with 3,6-diaminopyrazine-2,5-dicarboxylic acid (0.535 g, 2.70mmol), (1R,2S,3R,4R)-1-aminopentane-1,2,3,4,5-pentaol (0.978 g, 5.40mmol, 2.0 equiv.) and DMF (40 mL). To this was added triethylamine(0.546 g, 0.76 mL, 5.40 mmol, 2.0 equiv.) and PyBop (3.1 g, 5.94 mmol,2.2 equiv.). After an hour the reaction was complete by HPLC analysisand concentrated in vacuo keeping the temperature below 40° C. Themixture was taken up in water (10 mL), passed through a Sephadex G-10column and fractions containing a fluorescent product collected andlyophilized to obtain an impure solid. The target product,3,6-diamino-N²,N⁵-bis((1R,2S,3R,4R)-1,2,3,4,5-pentahydroxypentyl)pyrazine-2,5-dicarboxamide,was obtained by preparative C-18 RPHPLC: 160 mg, HRMS (theoretical)M+Na=547.1970; HRMS (Observed) M+Na=547.1969.

Example 19

Protocol for Assessing Renal Function

An example of an in vivo renal monitoring assembly 10 is shown in FIG. 2and includes a light source 12 and a data processing system 14. Thelight source 12 generally includes or is interconnected with anappropriate device for exposing at least a portion of a patient's bodyto light therefrom. Examples of appropriate devices that may beinterconnected with or be a part of the light source 12 include, but arenot limited to, catheters, endoscopes, fiber optics, ear clips, handbands, head bands, forehead sensors, surface coils, and finger probes.Indeed, any of a number of devices capable of emitting visible and/ornear infrared light of the light source may be employed in the renalmonitoring assembly 10. In one aspect, the light sources are LEDs whereone of the LEDs emits light near the absorbance maximum of the traceragent while the second LED emits light near the fluorescence emissionmaximum of the tracer agent. For example, one LED emits light at 450 nmwhile the second LED emits light at 560 nm.

Still referring to FIG. 2 , the data processing system 14 of the renalmonitoring assembly may be any appropriate system capable of detectingspectral energy and processing data indicative of the spectral energy.For instance, the data processing system 14 may include one or morelenses (e.g., to direct and/or focus spectral energy), one or morefilters (e.g., to fitter out undesired wavelengths of spectral energy),a photodiode or photomultiplier (e.g., to collect the spectral energyand convert the same into electrical signal indicative of the detectedspectral energy), an amplifier (e.g., to amplify electrical signal fromthe photodiode or photomultiplier), and a processing unit (e.g., toprocess the electrical signal from the photodiode or photomultiplier).The data processing system 14 is preferably configured to manipulatecollected spectral data and generate an intensity/time profile and/or aconcentration/time curve indicative of renal clearance of a pyrazinederivative of the present disclosure from patient 20. Indeed, the dataprocessing system 14 may be configured to generate appropriate renalfunction data by comparing differences in manners in which normal andimpaired cells remove the pyrazine derivative from the bloodstream, todetermine a rate or an accumulation of the pyrazine derivative in organsor tissues of the patient 20, and/or to provide tomographic images oforgans or tissues having the pyrazine derivative associated therewith.

By way of example and not limitation, in one aspect the system comprisestwo silicon photomultipliers. The first photomultiplier includes a longpass filter while the second photomultiplier is unfiltered. Thisarrangement permits both the fluorescence emission and diffusereflectance at the excitation and emission wavelengths to be measured.In one such embodiment, the fluorescence and diffuse reflectancemeasurement are combined into an Intrinsic Fluorescence measurement thatis compensated for variations in tissue optical properties. An exampleformula for combining the measurements is provided in Equation 1.

In one aspect for determining renal function, an effective amount of apyrazine derivative is administered to patients in need thereof (e.g.,in the form for a pharmaceutically acceptable composition). At least aportion of the body of the patient 20 is exposed to visible and/or nearinfrared light from the light source 12 as indicated by arrow 16. Forinstance, the light from the light source 12 may be delivered via afiber optic that is affixed to an ear of the patient 20. The patient maybe exposed to the light from the light source 12 before or afteradministration of the pyrazine derivative to the patient 20. In somecases, it may be beneficial to generate a background or baseline readingof light being emitted from the body of the patient 20 (due to exposureto the light from the light source 12) before administering the pyrazinederivative to the patient 20. When the pyrazine derivative that is inthe body of the patient 20 is exposed to the light from the light source12, the pyrazine derivative emanates light (indicated by arrow 18) thatis detected/collected by the data processing system 14. Initially,administration of the pyrazine derivative to the patient 20 generallyenables an initial spectral signal indicative of the initial content ofthe pyrazine derivative in the patient 20. The spectral signal thentends to decay as a function of time as the pyrazine derivative iscleared from the patient 20. This decay in the spectral signal as afunction of time is indicative of the patient's renal function.Additionally, if the tracer agent is injected into the vascular space ofa patient, the initial kinetics reflect the equilibration of the traceragent into the entire extracellular space of the patient. In someaspects, this equilibration is complete in less than 2 hours.

For example, in a first patient exhibiting healthy/normal renalfunction, the spectral signal may decay back to a baseline in a time ofT. However, a spectral signal indicative of a second patient exhibitingdeficient renal function may decay back to a baseline in a time of T+4hours. The extent of renal impairment or deficiency will affect thelength of time required for the signal to decay back to baseline. Agreater degree of renal impairment will require a longer period of time.As such, the patient 20 may be exposed to the light from the lightsource 12 for any amount of time appropriate for providing the desiredrenal function data. Likewise, the data processing system 14 may beallowed to collect/detect spectral energy for any amount of timeappropriate for providing the desired renal function data.

Additionally, GFR determination in a patient is not limited to a singledetermination based on a single administration of the tracer agent. Thetime between administration of the tracer agent and when it becomesundetectable in the patient may be subdivided into multiple smallersegments, and the GFR of the patient calculated for each smallersegment. In some aspects the time segments can overlap. By way ofexample and not limitation, if the entire time period before the traceragent become undetectable is 24 hours, then the time period can bedivided into four equal segments of six hours; each new time segmentbeginning at the end of the previous segment. In yet another aspect, thetime segments may overlap. For example, each individual time segment maybe four hours long, but a new time segment could begin every two hours.This would generate overlapping segments throughout the measurement. Tomore fully illustrate this nonlimiting example, if the tracer agent wasadministered at time equals 0 and became undetectable at time equals 24hours, then the following time segments may be generated: 0-4 hours, 2-6hours, 4-8 hours, 6-10 hours, 8-12 hours, 10-14 hours, 12-16 hours,14-18 hours, 16-20 hours, 18-22 hours, and 20-24 hours. The GFR of thepatient can be calculated in each time segment individually. This datawould them be used to more fully evaluate the health of the kidneys of apatient. The time segment may be any length that permits GFRdetermination and may be 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours or 12hours. Additionally, the time segments do not have to be identicalduring the measurement. The length of each time segment is selectedindividually without regard to any other time segment.

Pharmacokinetic Study Results

In a clinical study, 60 human patients were administered MB-102((2R,2′R)-2,2′-((3,6-diaminopyrazine-2,5-dicarbonyl)bis(azanediyl))bis(3-hydroxypropanoicacid)) prepared in Example 12) intravenously. Blood and urine werecollected in addition to the methods and techniques disclosed herein.Standard pharmacokinetic data was collected and comparison was madebetween the methods and techniques disclosed herein to Omnipaque®(iohexol), a known contrast agent used for GFR determination.

Shown in FIGS. 3A to 3D are data collected from the 60 human patientstested with MB-102. Plasma pharmacokinetic data was collected andanalyzed using methods known in the art and compared to the datameasured using the methods and techniques disclosed herein.

FIGS. 3A to 3D illustrate a two compartment pharmacokinetic model forthe elimination of MB-102. The model is consistent for patientsregardless of their GFR values. FIG. 3A is for patients having normalkidney function having a measured GFR (mGFR) of 120 mL/min. FIG. 3B isfor patients having a mGFR of 81 mL/min. FIG. 3C is for patients havinga mGFR of 28 mL/min. FIG. 3D is for patients having a mGFR of 25 mL/min.The first compartment in the two compartment model is the vascular totissue equilibrium while the second compartment illustrates renalexcretion only. On average, the time for equilibration is about onehour, and is subject dependent.

Shown in FIG. 4 is comparative data for the GFR measured usingOmnipaque® using traditional methods in comparison to MB-102, using themethods disclosed herein for patients having an eGFR ranging from about20 to about 140. The data shows a correlation coefficient of 0.97. Thisindicates that the method for determining patient GFR used hereinprovides similar results compared to known methods.

As part of the clinical 1 study for MB-102, urine was collected frompatients for 12 hours to determine the amount recovered and degree ofsecondary metabolism. As shown in FIG. 5 , for patients having an eGFRgreater than 60, greater than 99% of MB-102 was recovered unmetabolizedafter 12 hours. For patients having a mGFR value below 60, MB--102 alower percentage was recovered but it was likewise unmetabolized. Forpatients having normal, stage 1 and stage 2 renal function, 12 hourcollection time was sufficient for greater than 99% recovery of MB-102.In view of the pharmacokinetic data and the plasma half-lifedetermination, less than complete collection is readily understood forpatients having more serious renal function impairment.

Shown in FIG. 6 is the plasma concentration half-life of MB-102 forpatients having either normal kidney function, stage 1 renal impairment,stage 2 renal impairment, stage 3 renal impairment, or stage 4 renalimpairment. Based on this data, it is clear that in the urine collectionstudy above, longer than 24 hours will be required to clear all ofMB-102 from a patient's bloodstream. The normal renal function group hasan average plasma half-life of two hours, thus the 12 hour collectiontime is about 6 half-lives and is enough time to excrete most of theinjected dose. The stage 2 and 3 groups have an average plasma half-lifeof 2.5 hours, resulting in about 5 half-lives of excretion time which isalso enough to collect most of the injected dose. However, the 4 hourhalf-life for stage 3 and the 8 hour half-life for stage 4 does notallow all the injected dose to be collected in the 12 hour window forthe clinical study.

In a clinical study, the plasma pharmacokinetics was correlated as afunction of time (starting 2 hours after tracer agent injection andcontinuing to the end of the 12 hr study period) with the transdermalfluorescence pharmacokinetics measured on the sternum of the patients.High correlations between the plasma concentrations and fluorescenceintensity were observed for patients spanning a wide range of GFRvalues. Shown in FIGS. 7, 8 and 9 are three individual patients with GFRvalues between 23 mL/min/1.73 m² (stage 3b renal impairment) to 117mL/min/1.73 m² (normal kidney function). Thus, the plasmapharmacokinetics are correlated with the transdermal fluorescence forMB-102, for patients spanning a wide GFR range.

Transdermal GFR Determination (Fits to Full Data Set)

Kinetic analysis was done using LabView, Matlab, WinNonlin and MicrosoftExcel, version 2010, as follows. The transdermal fluorescence data werefit to a single exponential function between 2 hours (relative to thetime of injection, ensuring that the tracer agent has equilibratedacross the extracellular space) and the end of the available data(typically about 12 hours). For each subject two fits were performed:(1) with the offset fixed at zero, (2) with the offset allowed to vary.The exponential time constant determined from the fits is referred to asthe renal decay time constant (RDTC).

Linear regression, outlier exclusion, and calculation of the correlationcoefficient (R²) and standard error of calibration (SEC) were performedin Microsoft Excel, version 2010. The inverse of the RDTC was correlatedwith GFR using 4 different methods of GFR determination:

Un-normalized—the GFR, as determined from the plasma PK analysis of bothIohexol and MB-102.

BSA-normalized—the height and weight were used to estimate eachsubject's body surface area (BSA), according to method of Mosteller (NEngl J Med, 1987; 317(17)). The GFR determined by plasma PK analysis wasdivided by the ratio of the computed BSA to 1.73 m² (the BSA for a“standard” sized patient).

V_(d)-normalized (Method 1)—the GFR determined by plasma PK analysis wasdivided by the ratio of the volume of distribution (V_(d)) (alsodetermined from the PK analysis) to 14,760 mL, the V_(d) for a“standard” sized patient. The V_(d) for a standard-sized patient wasdetermined by forcing the average nGFR across all Group 1 patients to beequal for the V_(d) and BSA normalization methods. “nGFR” is used hereto refer to generalized methods (including both BSA and V_(d)) in whichGFR is normalized to body size.

V_(d)-normalized (Method 2)—a single exponential, with offset fixed atzero, was fit to the MB-102 plasma concentrations vs. time, between 2and 12 hours (relative to the time of tracer agent injection). Theinverse of the fitted time constant was multiplied by 14,760 mL (theV_(d) for a “standard” sized patient; see above), resulting in GFRnormalized by the volume of distribution.

Correlation coefficients (R²) and standard errors of calibration (SEC)for plots of plasma-derived GFR vs transcutaneous renal clearance rateare summarized in Table 2 and FIGS. 12 to 18 . In agreement withRabito's previous findings (J Nucl Med, 1993; 34(2): 199-207),normalization of the GFR by BSA increases R² and decreases SEC,confirming the hypothesis that the rate of renal clearance of MB-102provides a measure of the kidney efficiency that is independent of bodysize. Further, these results show that the volume of distribution(V_(d)) of the tracer agent is also effective for normalizing the GFR toa standard body size. As can be seen in the in Table 2, the BSA and thesecond V_(d), body size normalization methods were equally effectivewhen no outlier exclusion methods were applied, and the offset for theRDTC fits was fixed at zero.

TABLE 2 GFR SEC GFR norm. Offset Outlier Absolute Relative Compoundmethod Method Exclusions R² N (mL/min) (%) Iohexol none Fixed at 0 none0.6494 55 19.0 25.1% Iohexol BSA Fixed at 0 none 0.7804 55 13.5 19.1%Iohexol V_(d) Fixed at 0 none 0.7978 55 14.3 21.0% MB-102 none Fixed at0 none 0.6911 55 21.0 24.7% MB-102 BSA Fixed at 0 none 0.8242 55 13.718.0% MB-102 V_(d) (1) Fixed at 0 none 0.8016 55 15.8 20.1% MB-102 V_(d)(2) Fixed at 0 none 0.8211 55 14.1 19.3%

RDTC Fitting Offset Method

The transdermal fluorescence kinetics were fit by two different offsetmethods: (1) offset fixed at zero, and (2) offset allowed to vary. FIGS.18 and 19 show the resulting correlation plots for the fixed andvariable offset methods, respectively. Note that when the offset isfixed at zero (FIG. 18 ), the data is clustered more tightly in the lowGFR region of the correlation plot, whereas when the offset is allowedto vary (FIG. 19 ), the data clustering is tighter in the high GFRregion.

This observation points to instability in the baseline fluorescence. Inhealthy subjects with a high rate constant for renal clearance, thefluorescence agent was cleared before the end of the 12 hour datacollection period. In these subjects it was observed that the finalplateau level of the fluorescence did not always perfectly match theinitial pre-injection baseline fluorescence. Allowing the offset to varyin the fits for these healthy subjects allowed the fits to compensatefor this baseline uncertainty, thereby improving the reliability of theextracted clearance rate constant. However, in many subjects withcompromised kidney function, the agent was not fully cleared within the12 hour window in which the measurements were collected. In thesesubjects, including a variable offset in the fits was found to increasethe uncertainty in the fitted clearance rate constant. By fixing theoffset at zero, the reliability of the rate constant improved.

Based on these observations, a hybrid offset method was developed. Inthe hybrid method, if a fit with the offset fixed indicates that therate constant is high (i.e. healthy kidney function), then the offset isallowed to vary; otherwise (i.e. compromised kidney function) the offsetis fixed. The optimum transition point was selected to maximize the R²and minimize the SEC, as shown in FIG. 25 . The transition point shownon the x-axis is expressed as a time constant (in units of hours), whichis the inverse of the clearance rate constant. As can be seen in thefigure, the optimum time constant was in the range of 3.5 to 4.5 hours.A correlation plot employing the hybrid offset method (transition point:3.5 hours) is shown in FIG. 20 . A comparison of the fixed, variable,and hybrid offset methods, provided in Table 3, shows that the hybridoffset method results in substantial increase in R² and decrease in SECcompared to the other methods.

TABLE 3 GFR SEC GFR norm. Offset Outlier Absolute Relative Compoundmethod Method Exclusions R² N (mL/min) (%) MB-102 BSA Fixed at 0 none0.8242 55 13.7 18.0% MB-102 BSA Variable none 0.7721 55 15.7 38.0%MB-102 BSA Hybrid none 0.8278 55 13.6 16.8% MB-102 V_(d) (1) Fixed at 0none 0.8016 55 15.8 20.1% MB-102 V_(d) (1) Variable none 0.8554 55 13.539.3% MB-102 V_(d) (1) Hybrid none 0.9165 55 10.3 15.4% MB-102 V_(d) (2)Fixed at 0 none 0.8211 55 14.1 19.3% MB-102 V_(d) (2) Variable none0.8699 55 12.0 38.1% MB-102 V_(d) (2) Hybrid none 0.9199 55 9.4 14.5%

Data Exclusions

For some of the clinical subjects, the sensor did not remain fullyattached to the skin over the full 12 hours of the study. Reattachmentof the sensor post-injection often resulted in a significant shift inthe signal level. This could be due to: (1) inhomogeneity in the skinauto-fluorescence, (2) inhomogeneity in the interstitial fluid fractionin the skin, or (3) changes in coupling efficiency of the light into andout of the skin. To address this in the future, the sensor wasredesigned to have a smaller footprint and to be more adherent to thepatient. In order to make use of the clinical data, some data exclusionswere applied.

Five of the 60 clinical subjects were entirely excluded from thefluorescence kinetic analysis: (1) Subject 1 was excluded because thesensor repeatedly over-heated (in all subsequent subjects the maximumallowed blue LED power level was reduced by 60%), (2) Subjects 8, 37,and 49 were excluded because the sensor came fully off of the skin andthe original signal level could not be restored by re-attachment, (3)Subject 46 was excluded because the majority of the data was excludedduring the pre-processing step (the probable cause was an unintendedchange in the LED power or detector gain after tracer agent injection).

Different metrics for further excluding subject data were tested,including: (1) correlation coefficient between the IF and fit; (2) rootmean squared error (RMSE) of the IF relative to the fit; (3) thedifference between the RDTC determined from a one exponential and a twoexponential fit; (4) the signal-to-noise ratio, computed as theamplitude of the fitted exponential term divided by the RMSE; (5) thecoefficient of variation (CV) of the rate constant, when fitted overmultiple shorter time segments (e.g. 1-2 hours) within the full 12 hourdata set; (6) the estimated error of the rate constant fitted to the IF;and (7) the estimated error of the GFR determined from the plasma data.None of these metrics includes a priori information about thecorrelation coefficient or SEC.

In one aspect, using data exclusion method (6) above, the estimatederror of the rate constant fitted to the IF, divided by the rateconstant (expressed as a relative error), gave the results shown in FIG.26 . By excluding subjects for which the relative error of the rateconstant was greater than 1.4-1.8%, significant improvements in R² andSEC were observed. Choosing 1.75% as the cut-off metric, ten of the 55subjects were excluded. The resulting correlation plots are shown inFIGS. 21 and 22 .

This method was further applied as an exclusion metric to test whethererror in the plasma-derived GFR determinations are also contributing tothe data scatter in the correlation plots. FIG. 27 shows theoptimization of this metric. By selecting 5% as the cut-off foracceptability of the GFRs, a slight improvement in R² and SEC wasobserved. However, this necessitated the exclusion of an additional 10subjects, reducing the remaining subjects to 35. The resultingcorrelation plots are shown in FIGS. 23 and 24 . A numerical summary ofapplying the different data exclusion metrics is provided in Table 4.

TABLE 4 GFR Outlier SEC GFR norm. Offset Exclusion Absolute RelativeCompound method Method Methods R² N (mL/min) (%) MB-102 BSA Hybrid none0.8278 55 13.6 16.8% MB-102 BSA Hybrid (1) 0.8906 45 11.0 15.0% MB-102BSA Hybrid (2) 0.8716 35 12.0 14.5% MB-102 V_(d) (1) Hybrid none 0.916555 10.3 15.4% MB-102 V_(d) (1) Hybrid (1) 0.9575 45 7.4 14.1% MB-102V_(d) (1) Hybrid (2) 0.9614 35 7.0 10.2% MB-102 V_(d) (2) Hybrid none0.9199 55 9.4 14.5% MB-102 V_(d) (2) Hybrid (1) 0.9575 45 7.0 13.3%MB-102 V_(d) (2) Hybrid (2) 0.9597 35 7.7 9.6%

The calibration slope determined from the above-described methods can beapplied to generate transcutaneous measurements of body-size-correctedGFR (herein referred to as “tGFR”). FIG. 10 shows the correlationbetween the predicted and plasma GFR values with BSA normalization. Thecalibration accuracy can be depicted by plotting the tGFR against the“gold standard” determination of body-size-corrected GFR, derived fromthe plasma PK analysis. FIG. 10 shows the resulting correlation when theplasma-determined GFR values were normalized to patient body surfacearea (BSA), for which the correlation coefficient (R²) is 0.89.Employing the volume of distribution (V_(d)) to correct for body sizeinstead of BSA resulted in a substantially improved correlation of 0.96(FIG. 11 ).

When compared to the most commonly used method for estimating GFR inclinical practice, eGFR, these tGFR results demonstrate a potential forsubstantially improved accuracy. High accuracy is important in guidingclinical decisions. FIG. 28 and Table 1 illustrate the 5 stages ofchronic kidney disease (CKD). Misdiagnosis of the CKD may affect theclinical treatment course. A error grid plot was constructed to providevisualization of CKD misdiagnosis of GFR measurements relative to a goldstandard, as shown in FIGS. 29 a-c . Measurements falling within a boxwith all green borders are correctly classified by CKD stage.Measurements contained within yellow and green borders are misdiagnosedby one CKD stage. Measurements contained within red and yellow bordersare misdiagnosed by two CKD stages. FIG. 29 a shows the eGFR error gridfor the same group of subjects as was used in the above-describedanalysis. In this case, 2 of the 60 subjects were misdiagnosed by 2 CKDstages, 16 subjects were misdiagnosed by 1 stage; and 41 subjects werecorrectly diagnosed. Table 5 provides a summary of the CKD diagnosiserrors as a percent of total measurements. FIGS. 29 b and c provide theerror grid plots for transdermal GFR (tGFR) determination. Importantly,the tGFR error grids contain no misdiagnoses by 2 CKD stages. Further,tGFR by the V_(d) normalization method shows a substantial reduction in1-stage misdiagnoses, when compared to eGFR (see Table 5).

TABLE 5 % of Measurements by CKD Stage Diagnosis Error GFR Method 0 ±1±2 eGFR 70% 27% 3% tGFR, BSA norm. 71% 29% 0% tGFR, V_(d) norm. 84% 16%0%

Transdermal GFR Determination (Windowed Fits)

In the above example, the full available data sets (i.e. followingadministration of the tracer agent and equilibration, 10 hours offluorescence decay) were used for determining the GFR. This may beappropriate for patients with stable kidney function, but for patientswith or at risk of acute kidney injury, a more rapid and real-timerepeated assessment of GFR trend is needed. Even for patients withstable kidney function, waiting 12 hours for the GFR determination maybe inconvenient. Tables 6 and 7 show the results of varying theMeasurement Time Window and Single Injection Reporting Period for thesame group of subjects as already described above.

The Measurement Time Windows (column 1 in Tables 6 and 7) werenon-overlapping in this example, so that the Number of Windows (column 2in Tables 6 and 7) multiplied by the Measurement Time Windows indicatesthe total number of hours after equilibration over which GFR estimateswere made. The best results were obtained when the Measurement TimeWindow was long enough so that the fluorescence intensity decayssubstantially. The time needed to achieve the same fraction offluorescence intensity decay will vary according to the health of thekidney. Therefore, the offset was fixed at zero, except for the widestMeasurement Time Windows on subjects with healthy kidneys. The StandardError of Calibration is summarized for all subjects (column 5 in Tables6 and 7), as well as for subsets of subjects with nGFR below or above 75mL/min.

Using BSA normalized GFR as the reference comparison (Table 6), forsubjects with nGFR above 75, a Measurement Time Window of at least about1.5 hours was used in order to achieve an nGFR calibration accuracybelow 15 mL/min. For subjects with nGFR below 75, a Measurement TimeWindow of least about 3 hours was used in order to achieve nGFRcalibration accuracy below 10 mL/min. Using V_(d) normalized GFR as thereference comparison (Table 7), equivalent nGFR calibration accuracywere achieved with Measurement Time Windows of 0.5 hours and 2 hours,respectively. As can be seen in Tables 6 and 7, the calibration accuracytargets stated above were maintained across at least 2 non-overlappingMeasurement Time Windows. However, a significant increase in the SEC wastypically observed when the predictions were extended across 3 or 4Measurement Time Windows. By increasing (e.g. doubling or tripling) theMeasurement Window Time from that used for the first two GFRmeasurements, at least a third GFR measurement provided equivalentaccuracy. These results show the utility of providing an automaticallyadjusting Measurement Time Window, not just for accounting for differingSNR across different patients, but also to account for the diminishingSNR over time, as the tracer agent is progressively cleared by thekidneys.

TABLE 6 Meas. Num. Window Win- Offset SEC (mL/min/1.73 m²) (hrs) dowsMethod N All nGFR < 75 nGFR ≥ 75 0.5 1 Fixed 44 18.8 19.0 18.2 0.5 2Fixed 44 20.8 21.0 20.2 0.5 3 Fixed 44 21.0 20.8 21.5 0.5 4 Fixed 4423.4 20.9 27.5 1 1 Fixed 44 16.0 16.4 15.1 1 2 Fixed 44 17.3 16.0 19.6 13 Fixed 44 17.4 16.2 19.5 1 4 Fixed 44 19.5 16.1 24.8 1.5 1 Fixed 4512.0 11.2 13.3 1.5 2 Fixed 45 12.9 11.8 14.7 1.5 3 Fixed 45 17.4 12.424.0 1.5 4 Fixed 45 23.7 12.6 35.9 1.5 5 Fixed 45 28.5 18.3 41.1 1.5 6Fixed 45 41.0 20.4 63.0 2 1 Fixed 45 11.4 10.3 13.0 2 2 Fixed 45 13.711.6 16.8 2 3 Fixed 45 21.1 12.1 31.4 2 4 Fixed 45 28.2 19.2 39.7 3 1Fixed 45 10.7 9.4 12.7 3 2 Fixed 45 19.2 9.9 29.2 3 3 Fixed 45 29.6 16.544.4 4 1 Fixed 45 10.6 9.3 12.5 4 2 Fixed 45 53.3 11.4 88.0 5 1 Fixed 4510.6 8.4 13.6 5 2 Fixed 45 19.5 15.6 25.2 5 1 Variable 45 24.9 28.2 17.35 1 Hybrid 45 13.9 11.6 17.3 5 2 Hybrid 45 20.2 14.6 27.6 10 1 Fixed 4511.6 8.1 16.0 10 1 Variable 45 15.4 14.9 16.3 10 1 Hybrid 45 11.1 6.816.3

TABLE 7 Window Num. Length Win- Offset SEC (mL/min/1.476e4 mL) (hrs)dows Method N All nGFR < 75 nGFR ≥ 75 0.5 1 Fixed 44 17.4 19.5 11.5 0.52 Fixed 44 20.2 22.7 12.8 0.5 3 Fixed 44 20.4 21.9 16.8 0.5 4 Fixed 4422.1 21.8 22.8 1 1 Fixed 44 14.9 16.7 10.3 1 2 Fixed 44 15.6 15.6 15.4 13 Fixed 44 16.8 15.8 18.8 1 4 Fixed 44 19.5 15.7 25.8 1.5 1 Fixed 4510.4 11.5 8.0 1.5 2 Fixed 45 12.6 12.1 13.6 1.5 3 Fixed 45 17.2 12.624.0 1.5 4 Fixed 45 25.4 13.3 39.8 1.5 5 Fixed 45 29.7 18.8 44.1 1.5 6Fixed 45 41.1 20.7 65.0 2 1 Fixed 45 8.9 9.6 7.5 2 2 Fixed 45 14.0 10.818.9 2 3 Fixed 45 22.3 12.5 34.3 2 4 Fixed 45 30.7 20.2 44.9 3 1 Fixed45 8.9 8.8 9.1 3 2 Fixed 45 19.1 10.6 29.5 3 3 Fixed 45 32.3 17.9 50.0 41 Fixed 45 9.5 8.6 11.1 4 2 Fixed 45 53.4 12.4 90.9 5 1 Fixed 45 9.8 8.012.7 5 2 Fixed 45 20.8 16.7 27.3 5 1 Variable 45 23.2 27.3 11.5 5 1Hybrid 45 11.0 10.8 11.5 5 2 Hybrid 45 19.1 13.8 26.8 10 1 Fixed 45 12.49.0 17.3 10 1 Variable 45 11.6 13.0 7.9 10 1 Hybrid 45 7.0 6.6 7.9

Real-Time Transdermal GFR Measurement

The methods disclosed herein enable real-time transdermal GFRdetermination in patients. After intravascular injection of the traceragent into the subject, a waiting period of two hours was used to allowfor equilibration of the tracer agent into the extravascular space.After the two hour mark data was accumulated for one more hour beforeperforming a first fit to the RDTC. The first RDTC fit was performedwith the offset term fixed at zero. If the RDTC was less than 3.5 hoursthe fit was repeated, allowing the slope to vary, otherwise the originalRDTC (with fixed offset) was retained. The estimated error of the RDTCwas then divided by the RDTC. If the resulting relative error was lessthan 1.7%, the RDTC was converted into BSA-normalized GFR by invertingthe RDTC and multiplying by the slope shown in FIG. 23 ; otherwise theGFR was not reported. After the first RDTC fit, the fitting procedurewas repeated at approximately 3 second intervals, with the subsequentfits incorporating all of the data that was in the first fit as well asall data that had been subsequently accumulated (at time intervals ofabout one second). The same procedure was applied to the data collectedby two sensors: one placed over the manubrium of the sternum, and thesecond placed over the pectoralis major. The results generated inreal-time during the measurement are displayed in FIG. 30 . Over thefull course of the study, the agreement between the tGFR reported by thetwo sensors and the variation in the tGFR reported by each sensor waswithin 2 mL/min/1.73 m².

Stability Testing of MB-102

Samples of MB-102 were prepared and stored at 25° C. and 60% relativehumidity for 24 months. HPLC evaluation of each sample was performed atvarious time points to assess the stability of the samples. The resultsare shown in Table 8.

TABLE 8 Months Purity 0  >99% 6  >99% 12 98.9% 18 98.3% 24 97.8%

When introducing elements of the present disclosure or embodimentsthereof, the articles “a,” “an,” “the,” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising,”including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above processes and compositeswithout departing from the scope of the disclosure, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A method for determining a glomerular filtrationrate (GFR) in a human patient, said method comprising: administering tosaid patient a compound of Formula I, or a pharmaceutically acceptablesalt thereof, wherein the compound of Formula I is configured to emitspectral energy when exposed to electromagnetic radiation; transdermallymeasuring the spectral energy emitted by the compound of Formula I insaid patient over a measurement time window using a sensor attached on abody of said patient, the body comprising skin, wherein the spectralenergy is emitted by the compound of Formula I in response toelectromagnetic radiation delivered to the compound of Formula I in saidpatient; and determining, using a computing device in communication withthe sensor, the GFR in said patient based on the measured spectralenergy emitted by the compound of Formula I over the measurement timewindow, wherein the computing device determines the GFR by fitting anexponential function to the spectral energy as a function of time or alinear function to the log of the spectral energy as a function of timeto calculate a rate constant associated with renal clearance over themeasurement time window, wherein the rate constant is directly relatedto the GFR normalized to a body size metric of the patient; wherein inthe compound of Formula I,

each of X¹ and X² is independently —CO₂R¹, —CONR¹R², —CO(AA) or—CONH(PS); each of Y¹ and Y² is independently selected from the groupconsisting of —NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a single amino acid or apeptide chain comprising two or more amino acids, each amino acidselected from the group consisting of natural and unnatural amino acids,wherein the two or more amino acids of the peptide chain are linkedtogether by peptide or amide bonds and each instance of AA may be thesame or different than each other instance; PS is a sulfated ornon-sulfated polysaccharide chain comprising one or more monosaccharideunits connected by glycosidic linkages; and a is a number from 1 to 10,c is a number from 1 to 100, and each of m and n are independently anumber from 1 to
 3. 2. The method of claim 1, further comprisingdelivering electromagnetic radiation to the compound of Formula I insaid patient using the sensor attached on the body of said patient. 3.The method of claim 1, wherein the measurement time window is from about15 minutes to about 168 hours.
 4. The method of claim 1, wherein both X¹and X² are —CO(AA).
 5. The method of claim 4, wherein AA is D-serine, Y¹and Y² are each —NR¹R² and R¹═R² ═H.
 6. The method of claim 1, whereinthe administering is done via a single intravenous or transdermalinjection, and wherein the measuring is done via multiple measurementsafter the single injection, whereby determining the GFR is performed inreal time.
 7. The method of claim 1, further comprising measuring adiffuse reflectance of the skin prior to and/or after administering thecompound of Formula I.
 8. The method of claim 1, further comprisingrecording a baseline signal of fluorescence prior to administering thecompound of Formula I.
 9. The method of claim 1, further comprisingmonitoring equilibration of the compound of Formula I afteradministering the compound of Formula I.
 10. The method of claim 1,wherein the body size metric is body surface area.
 11. A system fordetermining a glomerular filtration rate (GFR) in a human patient, saidsystem comprising: an electromagnetic radiation source for deliveringelectromagnetic radiation to a compound of Formula I administered tosaid patient, wherein the compound of Formula I is configured to emitspectral energy when exposed to the electromagnetic radiation; a sensorfor transdermally measuring the spectral energy emitted by the compoundof Formula I in said patient over a measurement time window, the sensorconfigured to be attached on a body of said patient, the body comprisingskin; and a computing device configured to be connected in communicationwith the sensor, wherein the computing device is configured to determinethe GFR in said patient based on the spectral energy emitted by thecompound of Formula I and measured by the sensor over the measurementtime window, wherein the computing device is configured to determine theGFR by fitting an exponential function to the spectral energy as afunction of time or a linear function to the log of the spectral energyas a function of time to calculate a rate constant associated with renalclearance over the measurement time window, wherein the rate constant isdirectly related to the GFR normalized to a body size metric of thepatient; wherein in the compound of Formula I,

each of X¹ and X² is independently —CO₂R¹, —CONR¹R², —CO(AA) or—CONH(PS); each of Y¹ and Y² is independently selected from the groupconsisting of —NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a single amino acid or apeptide chain comprising two or more amino acids, each amino acidselected from the group consisting of natural and unnatural amino acids,wherein the two or more amino acids of the peptide chain are linkedtogether by peptide or amide bonds and each instance of AA may be thesame or different than each other instance; PS is a sulfated ornon-sulfated polysaccharide chain comprising one or more monosaccharideunits connected by glycosidic linkages; and a is a number from 1 to 10,c is a number from 1 to 100, and each of m and n are independently anumber from 1 to
 3. 12. The system of claim 11, wherein the sensorcomprises the electromagnetic radiation source, the sensor beingconfigured to generate and deliver the electromagnetic radiation to thecompound of Formula I administered to said patient.
 13. The system ofclaim 11, wherein the computing device is further configured todetermine a diffuse reflectance of the skin of said patient and, basedon the determined diffuse reflectance, cause at least one of an outputlevel of the electromagnetic radiation source and a detector gain levelof the sensor to be adjusted.
 14. The system of claim 11, wherein thecomputing device is further configured to record a baseline signalrepresentative of fluorescence being emitted from the body of saidpatient before the compound of Formula I is administered to saidpatient.
 15. The system of claim 11, wherein the computing device isfurther configured to determine that equilibration of the compound ofFormula I administered to said patient is complete and determine the GFRin said patient in response to determining that the equilibration iscomplete.
 16. A method for determining a glomerular filtration rate(GFR) in a human patient in real-time, said method comprising:administering to said patient a single injection of a compound ofFormula I, or a pharmaceutically acceptable salt thereof, wherein thecompound of Formula I is configured to emit spectral energy when exposedto electromagnetic radiation; transdermally measuring the spectralenergy emitted by the compound of Formula I in said patient overmeasurement time windows using a sensor attached on a body of saidpatient, the body comprising skin, wherein the spectral energy isemitted by the compound of Formula I in response to electromagneticradiation delivered to the compound of Formula I in said patient; anddetermining, using a computing device in communication with the sensor,the GFR in said patient based on the measured spectral energy emitted bythe compound of Formula I over each measurement time window, wherein,for each measurement time window, the computing device determines theGFR by fitting an exponential function to the spectral energy as afunction of time or a linear function to the log of the spectral energyas a function of time to calculate a rate constant associated with renalclearance over the respective measurement time window, wherein the rateconstant is directly related to the GFR normalized to a body size metricof the patient; wherein in the compound of Formula I,

each of X¹ and X² is independently —CO₂R¹, —CONR¹R², —CO(AA) or—CONH(PS); each of Y¹ and Y² is independently selected from the groupconsisting of —NR¹R² and

Z¹ is a single bond, —CR¹R²—, —O—, —NR¹—, —NCOR¹—, —S—, —SO—, or —SO₂—;each of R¹ to R² are independently selected from the group consisting ofH, —CH₂(CHOH)_(a)H, —CH₂(CHOH)_(a)CH₃, —CH₂(CHOH)_(a)CO₂H,—(CHCO₂H)_(a)CO₂H, —(CH₂CH₂O)_(c)H, —(CH₂CH₂O)_(c)CH₃, —(CH₂)_(a)SO₃H,—(CH₂)_(a)SO₃ ⁻, —(CH₂)_(a)SO₂H, —(CH₂)_(a)SO₂ ⁻, —(CH₂)_(a)NHSO₃H,—(CH₂)_(a)NHSO₃ ⁻, —(CH₂)_(a)NHSO₂H, —(CH₂)_(a)NHSO₂ ⁻, —(CH₂)_(a)PO₄H₃,—(CH₂)_(a)PO₄H₂ ⁻, —(CH₂)_(a)PO₄H²⁻, —(CH₂)_(a)PO₄ ³⁻, —(CH₂)_(a)PO₃H₂,—(CH₂)_(a)PO₃H⁻, and —(CH₂)_(a)PO₃ ²⁻; AA is a single amino acid or apeptide chain comprising two or more amino acids, each amino acidselected from the group consisting of natural and unnatural amino acids,wherein the two or more amino acids of the peptide chain are linkedtogether by peptide or amide bonds and each instance of AA may be thesame or different than each other instance; PS is a sulfated ornon-sulfated polysaccharide chain comprising one or more monosaccharideunits connected by glycosidic linkages; and a is a number from 1 to 10,c is a number from 1 to 100, and each of m and n are independently anumber from 1 to
 3. 17. The method of claim 16, wherein each of saidmeasurement time windows is from about 15 minutes to about 168 hours.18. The method of claim 16, wherein at least one pair of temporallyadjacent measurement time windows overlap.
 19. The method of claim 16,further comprising adjusting at least one of said measurement timewindows based on a quality metric associated with the transdermallymeasured spectral energy over the at least one of said measurement timewindows.
 20. The method of claim 16, further comprising monitoringequilibration of the compound of Formula I after administering thecompound of Formula I and prior to transdermally measuring the spectralenergy emitted by the compound of Formula I in said patient over a firstof said measurement time windows.